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Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

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CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus

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Lei Hana,1, Shuhua Mua,1, Zhendan Hea, Zhiwei Wangc, Junle Qud, Wencai Yeb,n, Jian Zhanga,nn a

School of Medicine, Shenzhen University, 518060 Shenzhen, PR China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, 510632 Guangzhou, PR China c Department of Neurosurgery, Shenzhen Shekou People's Hospital, 518060 Shenzhen, PR China d College of Optoelectronics Engineering, Shenzhen University, 518060 Shenzhen, PR China b

art i cle i nfo

ab st rac t

Article history:

6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) is a most commonly used antagonist of α-

Accepted 11 February 2016

amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the central nervous system. During the past two decades, studies had demonstrated that CNQX could

Keywords: CNQX

partially activate AMPA receptors that are located on the hippocampal and cerebellar interneurons, thus subsequently leading to the facilitation of inhibitory transmission. However, whether CNQX could enhance inhibitory synaptic transmission in the hypoglos-

AMPA Inhibitory transmission Patch-clamp Hypoglossal nucleus

sal nucleus remains elusive. Here, using whole-cell patch-clamp recording in the brainstem slice, we showed that CNQX greatly increased both frequency and amplitude of spontaneous inhibitory postsynaptic currents in the hypoglossal motoneurons, whereas D(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), N-methyl-D-aspartate (NMDA) receptor antagonist, had no effect on inhibitory synaptic transmission. Application of bicuculline and strychnine further identified that CNQX not only increased GABAergic sIPSCs but also glycinergic one in these motoneurons. Similar enhancement of inhibitory transmission was observed with application of DNQX, a quinoxaline derivative of CNQX, but not with application of GYKI 53655, a non-competitive antagonist of AMPA receptor. In the presence of tetradotoxin, the effect of CNQX on sIPSCs was abolished, suggesting that an increase in

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Abbreviations: CNQX, 6-cyano-7-nitroquinoxaline-2, 3-dione; ACSF,

artificial cerebrospinal fluid; AMPA,

methyl-4-isoxazolepropionic acid; D-AP5,

D-(-)-2-Amino-5-phosphonopentanoic acid; DNQX,

GABA,

hypoglossal motoneurons; NMDA,

gamma-aminobutyric acid; HMs,

α-amino-3-hydroxy-5-

6,7-dinitroquinoxaline-2,3-dione;

N-methyl-D-aspartate; TARP,

transmembrane AMPA

receptor regulatory protein; TTX, tetrodotoin n Q3 correspondence to: School of Medicine, Shenzhen University, Nanhai Ave 3688, 518060 Shenzhen, P.R. China.author. nn Corresponding author. E-mail addresses: [email protected] ( W. Ye), [email protected] ( J. Zhang). 1 These two authors contributed equally to this article. http://dx.doi.org/10.1016/j.brainres.2016.02.020 0006-8993/& 2016 Published by Elsevier B.V.

Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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presynaptic interneuron spike firing rate induced by CNQX was responsible for the facilitation of sIPSCs. Taken together, these results demonstrated that the excitatory effect of CNQX on presynaptic interneurons triggered enhancement of both GABAergic and glycinergic synaptic transmission within the rat hypoglossal nucleus. & 2016 Published by Elsevier B.V.

1.

Introduction

from the other brain regions (Marchetti et al., 2002; Rekling et al., 2000). These neurons received excitatory glutamatergic inputs

The hypoglossal nucleus, where the overwhelming majority of

from the primary motor cortex and local reticular formation as

neurons are cholinergic hypoglossal motoneurons (HMs) (Davidoff

well as inhibitory inputs from interneurons in the reticular

and Schulze, 1988; Viana et al., 1990), is suggested to provide a

formation adjacent to the hypoglossal nucleus (Li et al., 1996,

fundamental function in respiration, swallowing, suckling, and

1997). Most of these inhibitory inputs are glycinergic in nature,

mastication (Sawczuk and Mosier, 2001). By coupling with each

whereas the other inhibitory components are GABAergic (O'Brien

other through gap junction, HMs can directly innervate tongue

and Berger, 2001). Regulation of inhibitory synaptic transmission

muscles to accomplish the physiological functions through hypo-

can affect respiratory rhythm by altering HMs membrane potential

glossal nerve. Coordination in the activity of these coupled HMs can generate rhythmic signal to guide genioglossus muscle,

and changing the output pattern of HMs. Previously, studies had investigated the isolation of spontaneous

thereby is crucial for maintaining various functions in the upper

inhibitory neuronal activity within the central nervous system (CNS)

airway. The output (i.e., firing rates and burst patterns) of HMs can

in the presence of 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX),

be modulated by either excitatory or inhibitory inputs projected

an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)

Fig. 1 – CNQX-induced potentiation of inhibitory synaptic transmission in hypoglossal motoneurons. (A) Left panel: sample trace of sIPSCs from a HM before and after perfusion of CNQX (10 μM). Noted that CNQX exerted a robustly increased spontaneous events. Right panel: photomicrograph of a recorded HM in the left panel, as labeled with neurobiotin. (B) Cumulative probability plot of inter-event interval and amplitude of sIPSCs from a sample neuron. The inter-event interval curve significantly shifted leftward with application of CNQX, whereas the amplitude curve significantly shifted rightward with application of CNQX. (C) Bar graph shows the effect of CNQX on the frequency and amplitude of sIPSCs. (D) No detectable inward or outward basal current was found in HM treated with CNQX. (E) CNQX did not change membrane potential and input resistance of HM. Voltage deflections were responses to periodic current injections of
100 pA for 100 ms at 0.5 Hz. Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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receptor antagonist. It was found that blockade of AMPA receptors that are expressed on the local interneurons in this network was expected to hyperpolarize these interneurons, which may slightly attenuate the inhibitory neurotransmitters release from its axon terminals, without facilitating the release of these neurotransmitters. However, since 1990s, a number of studies had demonstrated that CNQX and its quinoxalinedione analog, 6,7-dinitroquinoxaline-2,3dione (DNQX), commonly used as antagonist of AMPA receptors, was demonstrated to partially activate these AMPA receptors inversely, leading to subsequent depolarization of GABAergic interneurons, hence more gamma-aminobutyric acid (GABA) was triggered to release from their axon terminals in the hippocampus and cerebellum (Brickley et al., 2001; Hashimoto et al., 2004; Maccaferri and Dingledine, 2002; McBain et al., 1992; Menuz et al., 2007). In these limited neural networks, CNQX and DNQX were considered as weak partial agonists of AMPA receptors but not as competitive antagonists. Similarly, a great enhancement of inhibitory synaptic transmission was found upon application of CNQX in the hypoglossal nucleus. It is essential and interesting to study the novel function of CNQX in inhibitory transmission within this neural circuitry.

perfusion of CNQX (10 μM) robustly enhanced spontaneous inhibitory synaptic transmission in most recorded HMs (n¼ 97/118, left panel; Fig. 1A). The rest of HMs exhibited little response to the perfusion of CNQX. After recording, HM was visualized by the treatment of neurobiotin (right panel, Fig. 1A). The neurobiotin-labeled HMs demonstrated several processes arising from its huge soma. Further analysis of the changes in both frequency and amplitude was conducted. With application of CNQX, the cumulative probability curve of inter-event interval significantly shifted leftward (po0.001) while that of the amplitude of sIPSCs significantly shifted rightward, indicating that CNQX induced enhancement in both frequency and amplitude(po0.001; Fig. 1B). Bar graph shows that CNQX significantly increased the mean frequency of sIPSCs to 309.3735.1% of that of the baseline level (n¼ 97, po0.001; Fig. 1C) and the mean amplitude of sIPSCs to 181.1718.2% of the baseline level (n¼ 97, po0.001; Fig. 1C). The increased frequency of sIPSCs strongly indicated that CNQX facilitated the release of inhibitory neurotransmitter from presynaptic terminals that project to the HMs. More-

2.

Results

2.1. CNQX-induced facilitation of inhibitory synaptic transmission within the hypoglossal nucleus

over, we investigated the effect of CNQX on the basal current and input resistance of HMs. Administration of CNQX did not elicit detectable inward or outward basal current in HMs (98.5712.5% of the control level, p40.05; n¼ 7; Fig. 1D). Similarly, there was no change in basal membrane potential

In order to verify whether CNQX could facilitate inhibitory synaptic transmission within the hypoglossal nucleus, we first performed whole-cell patch-clamp recordings in HMs with application of CNQX. Under voltage-clamp, bath

(control:


70.270.4 mV;

CNQX:


69.870.7 mV;

p40.05;

n¼ 6) and input resistance (98.373.5% of the control level, p40.05; n¼ 6) when application of CNQX. Sample trace was illustrated in Fig. 1E. This result implies that AMPA receptors

Fig. 2 – NMDA receptor was independent on CNQX-potentiated sIPSCs in the hypoglossal nucleus. (A) Sample traces of sIPSCs recorded in HMs. D-AP5 (50 μM) had no obvious effects on sIPSCs but greatly facilitated this inhibitory transmission when CNQX (10 μM) was further added into the bath solution. (B) Cumulative probability distributions of the inter-event intervals and amplitudes of the sIPSCs as shown in the Fig. A. With perfusion of D-AP5, no significant differences can be detected in the distributions of inter-event intervals and amplitudes except after application of CNQX. (C) Pooled results of the effects of DAP5 and subsequent added CNQX on the frequency and amplitude of sIPSCs. N.S.: not significant. Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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on the HMs are not involved in the facilitation of inhibitory synaptic transmission.

2.2. NMDA receptor was not involved in the CNQXtriggered facilitation of sIPSCs Contrary to the traditional view of blocking AMPA receptors, CNQX can directly activate AMPA receptors on inhibitory interneurons. This hypothesis has been verified in both the hippocampus and cerebellum. An alternative hypothesis is that CNQX blocks AMPA receptor-driving inhibitory interneurons impinging upon inhibitory premotoneurons to HMs, which was also expected to enhance inhibitory inputs to HMs. It is usually accepted that N-methyl-D-aspartate (NMDA) receptors are mostly co-localized with AMPA receptors on functional dendritic spines. Thus, blockade of NMDA receptors can be formulated to facilitate inhibitory transmission within the hypoglossal nucleus according to the second hypothesis. In fact, bath perfusion of D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5, 50 μM), an antagonist of NMDA receptors, was demonstrated little effect on inhibitory transmission(frequency, 86.273.0% of control level, n ¼8, p40.05; amplitude, 88.973.1% of control level, n¼ 8, p40.05; Fig. 2) but significantly enhance that after co-application of D-AP5 and CNQX in both frequency (286.9748.3% of control level, n¼ 8, po0.01; Fig. 2) and amplitude (179.9724.5% of control level, n¼ 8, po0.05; Fig. 2). The differential effects of D-AP5 and CNQX on inhibitory transmission within the hypoglossal nucleus may postulate that the facilitation of inhibitory transmission by CNQX was not likely to the result from disinhibition of inhibitory premotoneurons to HMs.

2.3. Both GABAergic and glycinergic synaptic transmission were facilitated by CNQX It is known that HMs receive both GABAergic and glycinergic inhibitory innervations from surrounding inhibitory interneurons. To better understand whether CNQX could facilitate GABAergic or glycinergic transmission within the hypoglossal nucleus, bicuculline (GABAA receptor antagonist) and strychnine (glycine receptor antagonist) were used in the present experiment, respectively. Administration of bicuculline (10 μM) completely eliminated CNQX-induced enhancement of sIPSCs with pre-application of strychnine (1 μM) in HMs, confirming that CNQX enhanced GABAergic synaptic transmission within the hypoglossal nucleus (Fig. 3A). In these experiments, CNQX (10 μM) significantly increased the frequency of GABAA receptor-mediated sIPSCs (293.6755.3% of the baseline value; n¼ 8, po0.05; Fig. 3A) but exerted no effects in amplitude (119.1717.0% of the baseline value, n¼ 8, p40.05; Fig. 3A). These results indicated that CNQX remarkably increased the frequency but had little effects on amplitude of GABAA receptor-mediated sIPSCs. In the hypoglossal nucleus, glycine and GABA are reported to be co-released onto both neonatal and juvenile HMs in a certain number of inhibitory terminals (O'Brien and Berger, 1999, 2001). Accordingly, perfusion of strychnine (1 μM) could also block CNQX-triggered facilitation of sIPSCs with pre-incubation of bicuculline (10 μM) in HMs, indicating that CNQX induced an enhancement of glycine receptor-mediated

sIPSCs in HMs. Statistical analysis showed that CNQX(10 μM) significantly increased the frequency of glycine receptormediated sIPSCs (381.7784.2% of the baseline value; n¼ 8, po0.05; Fig. 3B) and the amplitude (142.9711.8% of the baseline value; n¼ 8, po0.05; Fig. 3B).The analysis of kinetics in postsynaptic currents indicated that bicuculline-insensitive current decayed much more quickly than GABAA receptormediated current recorded at the same postnatal stage (Fig. 3C). The decay time of bicuculline-insensitive events was 7.270.4 ms, similar to that of glycine receptor-mediated current reported in HMs (O’Brien and Berger, 2001). On the other hand, bar graph showed that the decay time of GABAA receptor-mediated current was two folds longer than that of bicuculline-insensitive current (n¼ 8, po0.001, unpaired t test; Fig. 3C).All these results illustrated that CNQX not only induced enhancement of GABAA receptor-mediated sIPSCs but also triggered the facilitation of glycine receptormediated sIPSCs in HMs.

2.4. DNQX but not GYKI 53655 mimicked the effects of CNQX on sIPSCs We then tested the effects of quinoxaline derivative DNQX on the sIPSCs in HMs. DNQX (10 μM) also greatly enhanced inhibitory synaptic transmission in partial of recorded HMs (n¼ 9, Fig. 4A), in agreement with the results of CNQX-induced facilitation of sIPSCs in HMs. With application of DNQX, the frequency of sIPSCs was significantly increased (211.1729.0% of the control level; n¼ 9, po0.01; Fig. 4A) whereas the amplitude was also greatly increased (124.377.6% of the baseline; n¼ 9, po0.01; Fig. 4A). However, GYKI 53655, a non-competitive AMPA receptor antagonist, could not mimic the effect of CNQX on sIPSCs in all recorded HMs. GYKI 53655 (10 μM) had little effect on either frequency (82.2713.9% of the control level, n¼ 9, p40.05; Fig. 4B) or amplitude (97.478.7% of the control level, n¼ 9, p40.05; Fig. 4B) of sIPSCs in HMs. Further administration of CNQX (10 μM) still could not facilitate inhibitory synaptic transmission in the presence of GYKI 53655 (10 μM) (frequency, 79.179.9% of control level, n¼ 9, p40.05; amplitude, 85.479.0% of control level, n¼ 9, p40.05; Fig. 4B). These results suggest that only quinoxaline derivative AMPA receptor competitive antagonist could favor inhibitory transmitters release from inhibitory neurons that impinging on HMs while little effect with noncompetitive antagonist can be observed, which is in agreement with Menuz et al. (2007). In addition, it appears that blockade of AMPA receptors by non-competitive AMPA receptor antagonist in advance prevented activation of AMPA receptors by CNQX, hence subsequently abolished enhancement of sIPSCs within the hypoglossal nucleus.

2.5. Action potential-dependent facilitation of sIPSCs was induced by CNQX sIPSCs are composed of both action potential-dependent and action potential-independent components. To elucidate the effects of CNQX on the two subtypes of IPSCs, tetrodotoxin (TTX, 1 μM) was applied to eliminate action potential-dependent IPSCs. Bath administration of TTX abolished CNQX-induced enhancement of sIPSCs in HMs, afterwards, both frequency and amplitude of sIPSCs were significantly dropped to

Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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Fig. 3 – CNQX-induced facilitation of GABAergic and glycinergic synaptic transmission in the hypoglossal nucleus. (A) Top panel: representative traces showed that bicuculline (10 μM) completely eliminated CNQX-potentiated GABAergic sIPSCs in HM. strychnine (1 μM) was present in ACSF all the time. Middle panel: the cumulative probability distributions of the interevent intervals and amplitudes of the sIPSCs from the experiment as shown in the top panel of Fig. A. Bottom panel: pooled data demonstrates the effect of CNQX on the frequency and amplitude of GABAergic sIPSCs. All data were normalized to the control level. (B) Top panel: sample traces showed that strychnine completely eliminated CNQX-potentiated glycinergic sIPSCs in HM. Bicuculline was present in ACSF all the time. Middle panel: cumulative probability distributions of the interevent intervals and amplitudes of the sIPSCs from the experiment shown in the top panel of Fig. A. Bottom panel: pooled data demonstrates the of CNQX on frequency and amplitude of glycinergic sIPSCs. All data were normalized to the control level. (C) Left panel: deactivation time course of GABAA receptor-mediated current (black line) and glycine receptor-mediated current (grey line). They were averaged and normalized from representative postsynaptic events in the middle sample trace of Figs. A and B, respectively. The bicuculline-insensitive current decayed much quicker than GABAA receptor-mediated current, which is consistent with the bar graph (right panel). N.S.: not significant.

12.870.5% of the control level (n¼ 7, po0.05; Fig. 5A) and 52.574.6% of the control level (n¼7, po0.05; Fig. 5A), respectively. However, we could not exclude the possibility that CNQX might enhance the action potential-independent miniature IPSCs

(mIPSCs). To examine whether CNQX induces enhancement of mIPSCs, TTX was pretreated for 10 min to isolate the miniature events. Under this condition, it was found that CNQX failed to induce any enhancement of mIPSCs (n¼7, p40.05 in both

Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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Fig. 4 – DNQX greatly facilitated inhibitory transmission but GYKI 53655 had little effect. (A) Top panel: representative traces of sIPSCs recorded from a HM. DNQX (10 μM) mimicked CNQX-induced facilitation of sIPSCs. Middle panel: cumulative curve of inter-event interval and amplitude of sIPSCs from sample neuron in the top panel of the Fig. A. Significant difference was found in the distributions of inter-event intervals and amplitudes when perfused with DNQX. Bottom panel: pooled results demonstrate the effect of DNQX on the frequency and amplitude of sIPSCs. (B) Top panel: representative traces of sIPSCs recorded from a HM. GYKI 53655 (10 μM) was ineffective to either frequency or amplitude of sIPSCs (middle trace). Preadministration of GYKI 53655 prevented the facilitation of sIPSCs by CNQX (bottom trace). Middle panel: cumulative curve of inter-event interval and amplitude of sIPSCs from a sample neuron in top panel of the Fig. B. No significant differences were found in the distributions of inter-event intervals and amplitudes even treated with CNQX. Bottom panel: pooled results demonstrate the effect of GYKI 53665 and subsequent CNQX on the frequency and amplitude of sIPSCs. N.S.: not significant.

frequency and amplitude; Fig. 5B). These results suggest that the effect of CNQX is selective to action potential-dependent sIPSCs but not to mIPSCs. In other words, CNQX increased firing rate of presynaptic neurons via activation of AMPA receptors located on their presynaptic interneurons to enhance action potentialdependent release of inhibitory neurotransmitters onto adjoining HMs.

3.

Discussion

3.1. Facilitation of glycinergic transmission in the hypoglossal nucleus by CNQX Our electrophysiological studies demonstrated that CNQX and its quinoxaline derivative DNQX could dramatically enhance

inhibitory inputs to HMs. Consistent with earlier findings in the hippocampus and cerebellum, CNQX was found to enhance GABAergic transmission in HMs, however, it not only facilitated GABAergic transmission in HMs, but also exhibited its capability in enhancement of glycinergic transmission in HMs. This is the first report to show CNQX-potentiated glycinergic synaptic transmission in the CNS. The absence of CNQX-triggered enhancement of glycinergic transmission in the other brain regions may largely attribute to specific distribution patterns of functional glycine receptors in the CNS. Synaptic glycine receptors are rich in the retina, spinal cord and brainstem but rare in the neocortex and hippocampus, whereas GABAA receptors are widely distributed throughout the CNS (Lynch, 2004; Pirker et al., 2000). Furthermore, mixed GABAergic/glycinergic inhibitory synapses had been functionally characterized in motoneurons of the

Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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Fig. 5 – Action potential-dependent facilitation of sIPSCs triggered by CNQX. (A) Top panel: representative traces of sIPSCs recorded from a HM. TTX (1 μM) dramatically suppressed the frequency and amplitude of sIPSCs enhanced by CNQX. Middle panel: the cumulative probability distributions of inter-event intervals and amplitudes of the sIPSCs as shown in the top panel of Fig. A. TTX significantly shifted the cumulative curves of both inter-event intervals and amplitudes that were pre-treated with CNQX. Bottom panel: pooled results of the effect of TTX on sIPSCs were facilitated by CNQX. (B) Pre-perfusion of TTX prevented CNQX-induced enhancement of inhibitory transmission as illustrated in the sample traces (top panel). The distributions of cumulative probability of inter-event intervals and amplitudes of sIPSCs from the experiment as shown in the top panel of Fig. B (middle panel). Pooled data of the effects of CNQX on mIPSCs (bottom panel). N.S.: not significant.

hypoglossal nucleus (Muller et al., 2006; O'Brien and Berger, 2001). Accordingly, it is not strange that CNQX is able to facilitate both GABAergic and glycinergic synaptic transmissions within the hypoglossal nucleus. Given that the synaptic strength is fundamentally dependent on the three main factors: the numbers of synaptic contacts, size of postsynaptic depolarization/hyperpolarization caused by neurotransmitter release from a single synaptic vesicle (termed as quantal size), and probability of neurotransmitter release at each synapse (Del Castillo and Katz, 1956). With application of CNQX, the first two issues appeared to be constant while the probability of glycine/GABA release increased similarly to the control. Based on this theory, the amplitude of GABAergic currents should increase significantly as well as glycinergic ones with CNQX. Interestingly, CNQX only triggered significant increment in the amplitude of glycinergic sIPSCs but not in that of GABAergic sIPSCs. One possible interpretation for

this discrepancy is due to the differences in number of spare postsynaptic glycine/GABAA receptors in the control condition. The CNQX-induced rise in the amplitude of glycinergic currents is a result from the activation of a large number of spare postynaptic glycine receptors, whereas little increment in amplitude of GABAergic currents is due to the few spare postynaptic GABAA receptors in HMs. Morphological and electrophysiological evidences further demonstrated that functional glycine receptors are predominant over GABAA receptors at the postsynaptic sites, which is potentially in agreement with this interpretation.

3.2. Possible underlying mechanisms for activation of AMPA receptor Based on pharmacological and structural analysis, previous study had revealed that the presence of transmembrane

Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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AMPA receptor regulatory protein (TARP) auxiliary subunits could determine whether quinoxaline derivative acted as a partial agonist or a antagonist (Menuz et al., 2007). These accessory proteins play a crucial role not only in AMPA receptor trafficking (Chen et al., 2000; Kato et al., 2007; Soto et al., 2009; Tomita et al., 2003), but also in the regulation of synaptic targeting and gating as well as the pharmacological profiles of AMPARs (Bedoukian et al., 2006; Cho et al., 2007; Jackson and Nicoll, 2011; Milstein et al., 2007; Tomita et al., 2005). With the help of TARPs, the combination of CNQX to the corresponding site on AMPA receptors caused the opening of ion channel. This finding explained the "excitatory" role of CNQX on inhibitory transmission in the hippocampus and cerebellum as well as the hypoglossal nucleus in our present study. However, another study claimed that the shift of CNQX antagonism potency could be wholly attributed to the increased agonist potency caused by TARPs, which only fits for the equilibrium conditions but not for glutamatergic synapse (nonequilibrium conditions) (Maclean and Bowie, 2011). CNQX is regarded as a non-competitive antagonist at glutamatergic synapses as they concluded, however, it is impossible to explain our experimental results collected in the non-equilibrium conditions. In the present study, CNQX appeared to serve as a weak partial agonist of AMPA receptors. CNQX alone can evoke a macroscopic inward current (about 40 pA) in the cerebellum Golgi cells (Menuz et al., 2007), suggesting that at least in some AMPA receptors on inhibitory neurons, CNQX serves as a weak partial agonist through overcoming CNQX-elicited desensitization of AMPA receptor. It is known that most neurons in the CNS express TARPs that co-localized with AMPA receptors (Lein et al., 2007; Tomita et al., 2003). This distribution pattern of TARPs therefore raises an interesting question that why the "excitatory" action of CNQX is only observed in a limited number of brain regions. The possible explanation is that splice variant (flip/ flop) of AMPA receptors determines the extent of desensitization of AMPA receptors. The flop variants of AMPA receptors are less desensitized by their agonists (Bertolino et al., 1993; Geiger et al., 1995; Partin et al., 1994). Accordingly, inhibitory neurons dominant in the expression of flop variant of AMPA receptors could be depolarized by CNQX, which elicits larger inward currents than its diminishment by desensitization. In brainstem nuclei such as the hypoglossal nucleus, both splice variants in all subunits of AMPA receptors can be detected (Paarmann et al., 2000). Thus, it can be speculated that CNQXtriggered activation of flop variant of AMPA receptors that localized at inhibitory synapse is responsible for the "excitatory" action on inhibitory interneurons that innervate HMs. As to the other AMPA receptor agonists, previous studies showed a high degree of variability in the effect of DNQX on inhibitory transmission, although the excitatory action of CNQX is consistent among all the previous reports. Administration of DNQX, however, showed no effects on sIPSCs in the CA3 of the hippocampus (McBain et al., 1992), whereas in the cerebellum (Brickley et al., 2001) and the hypoglossal nucleus, DNQX exerted an excitatory effect. This different phenomenon may be due to lower sensitivity of DNQX to AMPA receptors as compared with CNQX, which needs further studies to elucidate the underlying mechanisms.

GYKI 53655, a structurally different AMPA receptor antagonist, produced negligible alterations in sIPSCs in HMs, suggesting that only quinoxaline related AMPA receptor antagonist could act as a partial agonist of AMPA receptors. Moreover, the similar action of GYKI 53655 on sIPSCs to that of D-AP5 further excluded the possibility of disinhibition of inhibitory pre-motoneurons that innervate HMs. Preblockade of AMPA receptors by this non-competitive AMPA receptor antagonist abolished facilitation of sIPSCs induced by CNQX, indicating that activation of AMPA receptors by CNQX was the only cause for the enhancement of inhibitory transmission within the hypoglossal nucleus.

3.3. Direct depolarization of inhibitory neurons by CNQX appears to be responsible for facilitation of sIPSCs Previous electrophysiological data showed a CNQX-triggered inward current in inhibitory cerebellar Golgi cells and Purkinje cells, indicating a direct depolarization of these cerebellar inhibitory neurons by CNQX (Menuz et al., 2007). Similarly, in the hippocampus, CNQX could induce depolarization, thereby subsequently increase the firing rate of inhibitory neurons but not of pyramidal neurons (Hashimoto et al., 2004; Maccaferri and Dingledine, 2002). Thus, it is reasonable that increased firing rate of inhibitory neurons can account for the enhancement of inhibitory synaptic transmission, as a result of action potentialdependent GABA/glycine release onto the postsynaptic receptors of HMs. Therefore, blockade of action potential by TTX prevented CNQX-potentiated inhibitory transmission in HMs (Fig. 5). To confirm a direct excitatory action of CNQX on AMPA receptors, further experiments should be conducted in the inhibitory interneurons that are impinged onto HMs.

3.4. Dual role of CNQX in the CNS and its possible problems In the present study, we observed that CNQX could serve as a weak partial agonist of AMPA receptors for potentiating inhibitory strength, although CNQX is widely used as an antagonist of AMPA receptors to eliminate AMPA current. Therefore, it should be cautious to use CNQX as a regular blocker of AMPA receptors in the study of neural circuits, especially in the hippocampus, cerebellum, and hypoglossal nucleus, due to its suggested dual role. In most cases, CNQX serves as a non-competitive blocker of AMPA receptors in glutamatergic synapses for eliminating phasic glutamatergic events, however, in a small population of inhibitory neurons, CNQX works as a weak partial agonist of the AMPA receptors to induce a tonic depolarizing currents. Thus, it is still pharmacologically feasible to use CNQX for isolating GABAA/glycine receptor-mediated currents, however, it should be careful during the in vitro or in vivo chronic application of CNQX as a long-lasting activator of AMPA receptors, since the subsequent over-excitation may lead to neuron death.

Please cite this article as: Han, L., et al., CNQX facilitates inhibitory synaptic transmission in rat hypoglossal nucleus. Brain Research (2016), http://dx.doi.org/10.1016/j.brainres.2016.02.020

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4.

Experimental procedure

Sprague Dawley (SD) rats of postnatal day (P) 4–17 were used in the experiments. All procedures and protocols on animal preparation were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80-23). In this study, all efforts were made to minimize the numbers and suffering of the animals to produce statistically significant data.

4.1.

Slice preparations

SD rats were first decapitated quickly after isoflurane inhalation and the brains were dissected out from the skull subsequently. The brainstem was then removed to ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 120 NaCl, 2 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 26 NaHCO3 and 11 glucose, saturated with 95% O2 and 5% CO2, pH 7.3. Coronal brainstem slices (300 μm), containing the hypoglossal nucleus, were cut by a vibratome from the brainstem. The freshly obtained brainstem slices were then transferred to a pre-warmed (33 1C) water bath incubator where glass incubation chamber contained oxygenated ASCF (pH 7.3) for about 1 h before recording. After incubation, the brainstem slice was transferred to the recording chamber equipped with infrared differential interference contrast (IR-DIC) optics (Olympus, Japan). HMs were identified from their characteristic location and morphology which can be distinguished from the neurons beyond the hypoglossal nucleus.

4.2.

Whole-cell patch-clamp recording

Patch electrodes were pulled from borosilicate glass (Harvard Apparatus, USA) using a P-97 micropipette puller (Sutter Instruments, USA). The pipette resistance was 3–6 MΩ for recording HMs. Under voltage-clamp mode, internal solution composed of (in mM) 140 CsCl, 2 MgCl2, 2 Na2ATP, 1 EGTA, and 10 HEPES, pH 7.3 (adjusted by CsOH), 290 mOsm, was used for recording spontaneous inhibitory postsynaptic currents. To generate CNQX-induced facilitation of sIPSCs, both AMPA and NMDA receptor antagonists were not delivered to the slices before recording. Slice in recording chamber was continuously perfused with oxygenated ACSF at a rate of 2 ml/min. An Ag–AgCl wire immersed to the bath was used as a reference electrode for grounding. All whole-cell currents were recorded at
70 mV. To measure input resistance of HM, periodic current injections of
100 pA for 100 ms at 0.5 Hz was employed in the experiment under currentclamp mode. The membrane potential was held around
70 mV. Voltage deflections were responses to periodic hyperpolarizing current. All data were collected by a multiclamp 700A amplifier, Digidata 1322A analog/digital interface board and Clampex 10.3 software (Axon Instruments, USA). These signals were filtered at 3 KHz and digitized at 10 KHz, and stored for subsequent analysis. Neuron was discarded if the access resistance was over 20 MΩ or changed by more than 15% after recording. All recordings were performed at room temperature (20–25 1C).

4.3.

Morphology

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1132 1133 1134 To visualize the morphology of the whole-cell recorded HMs, 1135 the pipette was filled with freshly prepared 0.3% of neuro1136 biotin (Vector, USA) in CsCl based-internal solution. After 1137 recording for at least more than 30 min, the slice was 1138 transferred to 4% paraformaldehyde fixative in 0.1 M PB and 1139 immersed overnight at41C.After rinsing with PBS for 3 times 1140 (each 10 min), the section was incubated in avidin conjugated 1141 with Texas red (Molecular Probes, 1:1,000 diluted by 0.3% 1142 Triton-PBS) for at least 4 h in room temperature. Finally, the 1143 reaction was stopped by rinsing the section with PBS (10 min) 1144 for 3times. The section was then mounted on gelatin-coated 1145 slide for further observation. Digital images were captured by 1146 a fluorescent microscope (Axioplan II, Zeiss, Germany) with 1147 CCD camera (Spot, Diagnostic Instrument, USA). 1148 1149 4.4. Drugs 1150 1151 The following drugs were obtained from (1) Sigma chemical 1152 company (Sigma-Aldrich, USA): strychnine and TTX; (2) Tocris 1153 Bioscience: CNQX, DNQX, D-AP5, bicuculline, and GIKI 53655. 1154 Drugs were dissolved as stock solutions in distilled water or 1155 DMSO. The final concentration of DMSO was less than 0.1%. All 1156 stock solution of the applied drugs was freshly added to ACSF on 1157 the day of experiment. Drugs were delivered by a gravity-feed 1158 system connected to the bath-recording chamber. 1159 1160 4.5. Data analysis 1161 1162 All data were processed off-line in Mini Analysis 6.0.3 1163 (Synaptosoft, USA), Clampfit 10.3 (Axon Instruments, USA), 1164 and SigmaPlot 12.0 (Jandel Scientific, Germany) software. 1165 IPSCs were analyzed by Mini Analysis program. Only single 1166 peak events were chosen for kinetic analysis. Numerical data 1167 were presented as the mean 7S.E.M. Statistical comparison 1168 of two cumulative probabilities was done by a Kolmogorov1169 Smirnov test. All the other comparisons were assessed using 1170 paired or unpaired Student's t-tests as appropriate. po0.05 1171 * was considered as significant difference. symbolizes po0.05, 1172 ** *** represents for po0.01 and represents for po0.001. 1173 1174 1175 Acknowledgments 1176 This work was supported by National Natural Science Foun- Q5 1177 1178 dation of China (81400064). 1179 1180 r e f e r e nc e s 1181 1182 1183 Bedoukian, M.A., Weeks, A.M., Partin, K.M., 2006. Different 1184 domains of the AMPA receptor direct stargazin-mediated trafficking and stargazin-mediated modulation of kinetics. J. 1185 Biol. Chem. 281, 23908–23921. 1186 Bertolino, M., Baraldi, M., Parenti, C., Braghiroli, D., DiBella, M., 1187 Vicini, S., Costa, E., 1993. Modulation of AMPA/kainate recep1188 tors by analogues of diazoxide and cyclothiazide in thin slices 1189 of rat hippocampus. Recept. Channels 1, 267–278. 1190 Brickley, S.G., Farrant, M., Swanson, G.T., Cull-Candy, S.G., 2001. 1191 CNQX increases GABA-mediated synaptic transmission in the

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