Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius.

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Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius Yoshiaki Ohi, Satoko Kimura, Akira Hajin Laboratory of Neuropharmacology, School of Pharmacy, Aichi Gakuin University, Nagoya, Japan

art i cle i nfo

ab st rac t

Article history:

Activity of second-order relay neurons in the nucleus tractus solitarius (NTS) is regulated

Accepted 23 April 2014

by peripheral and intrinsic synaptic inputs, and modulation of those inputs by metabotropic glutamate receptors (mGluRs) has been proposed. This study investigated effects of mGluR activation on glutamatergic transmission in the NTS second-order neurons of

Keywords: Metabotropic glutamate receptors Excitatory postsynaptic currents Presynaptic modulation Nucleus tractus solitaries Guinea pig

guinea pigs. Whole-cell patch-clamp recordings from the brainstem slices revealed that activation of mGluRs exerted its effects on the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) but not on the amplitude. The sEPSC frequency was increased by an agonist of group I mGluRs, and it was decreased by an mGluR1 antagonist but not by an mGluR5 antagonist. The agonists of group II and III mGluRs decreased the sEPSC frequency, while their antagonists alone had no effect. Perfusion of cystine or TBOA, either of which elevates extracellular glutamate concentration, resulted in an increase in the sEPSC frequency, leaving the amplitude unchanged. The increased frequency of sEPSCs was returned to control by an mGluR1 antagonist. The tractus solitarius-evoked EPSCs were not altered by an agonist of group I mGluRs, whereas they were decreased along with an increase in paired-pulse ratio by agonists of group II and III mGluRs. These results suggest that mGluRs are present at the presynaptic sites in the NTS second-order neurons in guinea pigs. The mGluR1s function to facilitate the release of glutamate from axon terminals of intrinsic interneurons and the group II and III mGluRs play an inhibitory role in glutamatergic transmission. & 2014 Published by Elsevier B.V.

Abbreviations: EPSC,

excitatory postsynaptic current; mGluR,

metabotropic glutamate receptor; NTS,

nucleus tractus solitarius;

TBOA, DL-threo-β-benzyloxyaspartate n Correspondence to: Laboratory of Neuropharmacology, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan. Fax: þ81 52 757 6799. E-mail addresses: [email protected] (Y. Ohi), [email protected] (S. Kimura), [email protected] (A. Haji). http://dx.doi.org/10.1016/j.brainres.2014.04.031 0006-8993/& 2014 Published by Elsevier B.V.

Please cite this article as: Ohi, Y., et al., Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius. Brain Research (2014), http://dx.doi.org/ 10.1016/j.brainres.2014.04.031

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1.

Introduction

The nucleus tractus solitarius (NTS) situated in the dorsal medulla oblongata is the relay nucleus of autonomic nervous systems (Andresen and Kunze, 1994), in which the visceral sensory afferents primarily synapse onto the second-order neurons. In addition to the peripheral inputs, these neurons receive projections from the intrinsic local network that maintains and/or modulates the neuronal excitability in rats (Champagnat et al., 1986; Fortin and Champagnat, 1993; Kawai and Senba, 1996) and in guinea pigs (Haji and Ohi, 2010; Ohi et al., 2007, 2011). The excitatory synaptic transmission from both peripheral and intrinsic sources is mainly carried by glutamate through ionotropic receptors (Andresen and Yang, 1990). It is generally accepted that ionotropic glutamate receptors function in the fast-acting neurotransmission, while metabotoropic glutamate receptors (mGluRs) modify it in the central nervous system (Cartmell and Schoepp, 2000; Conn and Pin, 1997; Schoepp, 2001). There have been organized into three mGluR groups based on sequence homology, second messenger system involvement and pharmacology. The group I mGluRs (mGluR1 and 5) are distributed at the postsynaptic membrane and provide an excitatory modulation through activation of Gq proteins. The group II (mGluR2 and 3) and III (mGluR4, 6, 7 and 8) mGluRs are located at the presynaptic terminals and act as autoreceptors that inhibit glutamate release via activation of Gi/o proteins. In the NTS, mRNA expression of all eight subtypes of mGluRs and protein expression of mGluR1, 2, 3, 5 and 7 are reported (Austgen et al., 2009; Hay et al., 1999; Hoang and Hay, 2001). It has been already demonstrated in the rat NTS neurons that the group II and III mGluRs play an inhibitory regulation of glutamate

release from intrinsic interneurons (Fernandes et al., 2011) and from peripheral afferent terminals (Chen et al., 2002). On the other hand, activation of the group I mGluRs failed to alter the glutamatergic transmission in the NTS neurons (Chen et al., 2002; Fernandes et al., 2011). However, there are some incongruous findings in other central areas. For example, the presynaptic localization of group I mGluRs and enhancement of glutamate release by their activation have been shown in the cortex (Moroni et al., 1998; Musante et al., 2008) and the spinal trigeminal nucleus (Song et al., 2009). The mGluR1 and 5 were suggested to be involved in the integration of pulmonary afferent information in the NTS (Hay et al., 1999), since dense immunoreactivities of mGluR1 and 5 were shown in the ventrolateral, interstitial and commissural subnuclei of NTS which receive projection from respiratory afferents originating sensory endings in the trachea, larynx and bronchi (Kalia and Mesulam, 1980). Furthermore, we have revealed that centrally-acting antitussives inhibited the glutamatergic transmission in the guinea pig NTS neurons (Haji and Ohi, 2010; Ohi et al., 2007, 2011). Together, there is a possibility that these antitussives may interact with the mGluR-mediated modulation of synaptic transmission. Therefore, it is of particular importance whether glutamate can be a slow metabotropic transmitter to control its own release in NTS neurons. The present study was undertaken to investigate the effects of mGluR activation on the tractus solitarius (TS)-evoked and spontaneously occurring excitatory postsynaptic currents (eEPSCs and sEPSCs, respectively) in second-order neurons using a slice patch-clamp technique. We used the guinea pig brainstem slice, because this animal, but not the rat, is suitable for respiratory-related experiments such as cough (Belvisi and Bolser, 2002; Ohi et al., 2004, 2005) and because no data on mGluRs in the guinea pig NTS has been reported.

Fig. 1 – Effects of mGluR agonists on the sEPSCs in second-order NTS neurons. Traces of sEPSCs taken during before (control) and 4 min after the onset of DHPG (a group I mGluR agonist, 10 μM, A1), DCG IV (a group II mGluR agonist, 10 μM, B1) or L-AP4 (a group III mGluR agonist, 30 μM, C1). (A2, B2, C2) Relative values of the amplitude and frequency of sEPSCs. Values are the mean7SEM (n ¼6). nPo0.05, nnPo0.01 vs. control (paired t-test). Please cite this article as: Ohi, Y., et al., Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius. Brain Research (2014), http://dx.doi.org/ 10.1016/j.brainres.2014.04.031

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2.

Results

A total of 85 NTS second-order neurons were used in the present study. All recordings were made in the presence of picrotoxin (100 μM). These neurons showed monosynaptic EPSCs with a short latency (3.871.5 ms, n¼ 85) and small jitter (73.973.0 μs, n ¼85) from stimulus onset in response to electrical stimulation of the TS.

2.1. Effects of mGluR agonists on spontaneously occurring EPSCs Fifty-five out of 85 neurons exhibited stable sEPSCs. In control, the mean amplitude and frequency of sEPSCs were 14.273.2 pA and 7.372.1 Hz (n¼ 55), respectively. The sEPSCs were completely abolished by CNQX (n ¼5, data not shown), indicating that the neurons analyzed here receive

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predominantly glutamatergic synaptic inputs through activation of non-N-methyl-D-aspartate (non-NMDA) or 2-amino-3(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptors. No spontaneous inhibitory postsynaptic currents were recorded with picrotoxin (100 μM). Fig. 1 illustrates representative traces of sEPSCs during control and perfusion of each agonist of three mGluR groups. All agonists exerted their effects on only the sEPSC frequency while sparing the amplitude. DHPG (an agonist of group I mGluRs, 10 μM) increased the frequency of sEPCSs (228749% of control, n¼ 6, Po0.05), but it had no significant effect on their amplitude (11878% of control, P40.05). DCG IV (an agonist of group II mGluRs, 10 μM) decreased the sEPSC frequency (6277% of control, n¼ 6, Po0.05) without effect on the amplitude (9878% of control, P40.05). L-AP4 (an agonist of group III mGluRs, 30 μM) decreased the sEPSC frequency (7274% of control, n ¼6, Po0.01) with no change in the amplitude (9573% of control, P40.05). In all cases

Fig. 2 – (A1) Effects of antagonists of group I mGluRs on the sEPSCs in second-order NTS neurons. Traces of sEPSCs taken during before (control) and 4 min after the onset of LY367385 (an mGluR1 antagonist, 100 μM) or MPEP (an mGluR5 antagonist, 10 μM). (A2) Effects of antagonists of group II and III mGluRs on the sEPSCs in second-order NTS neurons. Traces of sEPSCs taken during before (control) and 4 min after the onset of LY341495 (a group II mGluR antagonist, 1 μM) or CPPG (a group III mGluR antagonist, 200 μM). (B) Relative values of the amplitude and frequency of sEPSCs. Values are the mean7SEM (n ¼ 5). nn Po0.01 vs. control (paired t-test). Please cite this article as: Ohi, Y., et al., Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius. Brain Research (2014), http://dx.doi.org/ 10.1016/j.brainres.2014.04.031

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using either agonist, the sEPSCs recovered to the control level 5 min after the washout.

2.2. Effects of mGluR antagonists on spontaneously occurring EPSCs The effects of selective antagonists of mGluRs on sEPSCs were examined (Fig. 2). LY367385 (an antagonist of mGluR1s, 100 μM) decreased the sEPSC frequency (5073% of control, n¼ 5, Po0.01) without effect on the amplitude (8377% of control, P40.05), while MPEP (an antagonist of mGluR5s, 10 μM) had no significant effect on sEPSCs (Fig. 2B). Neither

antagonist of group II (LY341495, 1 μM) nor group III mGluRs (CPPG, 200 μM) had any discernible effects on sEPSCs (Fig. 2B). Antagonism by LY367385 against the DHPG-induced facilitation of sEPSC frequency was investigated (Fig. 3). Drugs were applied sequentially to single cells by a following order; DHPG, DHPGþLY367385 and DHPGþMPEP. Again, application of DHPG (10 μM) significantly increased the sEPSC frequency. The plots of cumulative probabilities of sEPSCs clearly show that the distribution curve of the inter-event interval was shifted to the left side and that of the amplitude was unchanged (Fig. 3B). The increase in sEPSC frequency induced by DHPG was returned to the nearly control level by an additional application of LY367385 (100 μM, 115713% of

Fig. 3 – (A) Antagonism of LY367385 or MPEP against DHPG-induced facilitation of sEPSCs in a second-order NTS neuron. Drugs were applied sequentially. Traces of sEPSCs taken before (control), during DHPG (10 μM) and co-perfusion of LY367385 (DHPGþLY367385 100 μM) or MPEP (DHPGþMPEP 10 μM). (B) Cumulative probability of the amplitude and inter-event interval (IEI) of sEPSCs for the neuron in (A). The distribution of IEI was significantly shifted to the leftward by DHPG (Po0.01 vs. control, Kolmogorov–Smirnov test), but that of the amplitude was unchanged (P40.05 vs. control, Kolmogorov–Smirnov test). (C) Relative values of the amplitude and frequency of sEPSCs. Values are the mean7SEM (n¼ 5). nPo0.05 vs. control (paired ttest). ♯Po0.05, N.S.; not significant (ANOVA followed by Bonferroni corrected multiple t-test). Please cite this article as: Ohi, Y., et al., Modulation of glutamatergic transmission by metabotropic glutamate receptor activation in second-order neurons of the guinea pig nucleus tractus solitarius. Brain Research (2014), http://dx.doi.org/ 10.1016/j.brainres.2014.04.031


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control, n¼ 5, Po0.05 vs. DHPG), but it was not affected by MPEP (10 μM, 193731% of control, n¼ 5, P40.05 vs. DHPG). Taken together, it seems likely that mGluR1s primarily modulate glutamatergic transmission under our recording conditions.

2.3. Effects of group I mGluR antagonists on spontaneously occurring EPSCs in condition where the extracellular glutamate concentration was elevated Cystine activates cystine/glutamate exchanger in the membrane, resulting in rise in the extracellular glutamate concentration (Oldenziel et al., 2007). Fig. 4 illustrates a typical response of sEPSCs during perfusion of cystine. Drugs were applied sequentially to single cells by a following order; cystine, cystineþLY367385 and cystineþMPEP. Cystine (30 μM) increased the sEPSC frequency (12774% of control, n¼ 9, Po0.05), leaving the amplitude unchanged (10076% of control, P40.05). The cystine-induced facilitation of sEPSC frequency was diminished by an additional application of LY367385 (100 μM, 102711% of control, n¼ 9, Po0.05 vs. cystine) but not by MPEP (10 μM, 141712% of control, n ¼9, P40.05 vs. cystine). TBOA inhibits excitatory amino acid transporters (EAATs) and accumulates the glutamate content in the synaptic cleft (Oldenziel et al., 2007). Drugs were applied sequentially to single cells by a following order; TBOA, TBOAþLY367385 and TBOAþMPEP. Perfusion of TBOA (100 μM) increased only the sEPSC frequency (163719% of control, n¼ 7, Po0.05, Fig. 5) without effect on the amplitude (113713% of control, P40.05). The TBOA-induced increase in sEPSC frequency was abolished by LY367385 (100 μM, 105720% of control, n¼ 7, Po0.05 vs. TBOA) but not by MPEP (10 μM, 142725% of control, n¼ 7, P40.05 vs. TBOA). In either case with cystine or TBOA application, LY367385 did not lower the sEPSC frequency below the control level, suggestive of no activation of the group II and III mGluRs during cystine or TBOA application.

2.4.

Effects of mGluR agonists on TS-evoked EPSCs

Effects of the mGluR activation on glutamatergic transmission from the peripheral afferent terminals were investigated in 30 second-order neurons. All recordings were made in the presence of picrotoxin (100 μM). The TS-induced sEPSCs were completely abolished by CNQX (10 μM) application in five neurons tested (data not shown). Polysynaptic inhibitory currents were not observed with picrotoxin (100 μM). The mean resting membrane potential was 62.774.1 mV (n¼ 30). Fig. 6 shows effects of each agonist of mGluRs on two serial eEPSCs induced by double stimulus pulses in secondorder neurons. Perfusion of DHPG (10 μM) had neither effect on the peak amplitude of the first eEPSC (173719 pA for control vs. 172714 pA for DHPG, n¼5, P40.05) nor the pairedpulse ratio (PPR; 0.5770.11 for control vs. 0.6670.10 for DHPG, n¼ 5, P40.05) of responses following paired TS stimuli. DCG IV (10 μM) significantly decreased the peak amplitude of the first eEPSC (182719 pA for control vs. 92714 pA for DCG IV, n¼ 6, Po0.01). It had a stronger effect on the first eEPSC than

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the second one, leading to a marked increase in the PPR (0.8570.11 for control vs. 1.3470.21 for DCG IV, n ¼6, Po0.05). L-AP4 (30 μM) decreased the peak amplitude of the first eEPSC (17078 pA for control vs. 96711 pA for L-AP4, n¼ 6, Po0.01) and increased the PPR (0.7370.02 for control vs. 1.1570.11 for L-AP4, n¼ 6, Po0.01). These effects of each agonist of group II and III mGluRs disappeared 5 min after the washout. Neither drug induced any apparent shift of the baseline current, suggesting that these mGluR agonists do not influence the postsynaptic membrane conductance.

3.

Discussion

Activation of mGluRs influences neuronal processes in the brain by a variety of different pathways, notably employing as intermediary a rise in intracellular Ca2þ concentration through Gq proteins for the group I mGluRs or a fall in cAMP concentration through Gi/o proteins for the group II and III mGluRs (Cartmell and Schoepp, 2000; Schoepp, 2001). The final result may be to modify neuronal excitability or to alter the efficacy of synaptic transmission either pre- or postsynaptically. The mGluRs have a high affinity for glutamate, thus opening the possibility of modulation by glutamate released from the peripheral afferents or intrinsic axon terminals. The goal of the present study was to provide evidence of the presence and physiological function of mGluRs in the NTS second-order neurons of guinea pigs by using their selective agonists and antagonists. To avoid underestimation of their effects on glutamatergic transmission, relatively higher or sufficient concentrations were employed in this study, according to the previous reports (Chen et al., 2002; Fernandes et al., 2011; Kingston et al., 1998, 2002; Morishita and Alger, 2000). Therefore, although only one concentration was used for each agent, the results obtained here are thought to be reliable. The major findings are that: (1) the group I mGluRs may not be located at the synapses connected with TS-afferent terminals, (2) the mGluR1s, but not mGluR5s, play a facilitatory regulation of glutamate release at the presynaptic terminals projecting from intrinsic local circuits, and (3) the group II and III mGluRs are present in both TS-afferent and intrinsic presynaptic terminals, and provide an inhibitory regulation.

3.1. Modulation of NTS glutamatergic transmission by group I mGluRs In the guinea pig NTS second-order neurons, the amplitude of TS-evoked EPSCs was unchanged during activation of the group I mGluRs by DHPG (10 μM). The apparent lack of effect of the agonist bears out the findings in the rat NTS neurons (Chen et al., 2002) and other neural networks (Conn and Pin, 1997; Pin and Duvoisin, 1995). The present result suggests that the group I mGluRs do not regulate glutamatergic transmission from the TS-afferent terminals. On the other hand, there is a recent report that activation of the presynaptic group I mGluRs on retinal terminals can suppress the retinogeniculate glutamatergic transmission to the dorsal lateral geniculate nucleus of the thalamus (Lam and Sherman, 2013). Since a very high concentration of DHPG


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Fig. 4 – (A) Antagonism of LY367385 or MPEP against cystine-induced facilitation of sEPSCs in a second-order NTS neuron. Drugs were applied sequentially. Traces of sEPSCs taken before (control), during cystine (30 μM) and co-perfusion of LY367385 (cystineþLY367385 100 μM) or MPEP (cystineþMPEP 10 μM). (B) Cumulative probability of the amplitude and inter-event interval (IEI) of sEPSCs for the neuron in (A). The distribution of IEI was significantly shifted to the leftward by cystine (Po0.01 vs. control, Kolmogorov–Smirnov test), but that of the amplitude was unchanged (P40.05 vs. control, Kolmogorov–Smirnov test). (C) Relative values of the amplitude and frequency of sEPSCs. Values are the mean7SEM (n ¼ 9). nPo0.05, nnPo0.01 vs. control (paired t-test). ♯Po0.05, N.S.; not significant (ANOVA followed by Bonferroni corrected multiple t-test).

(125 μM) was used there, it is difficult to compare simply such results with ours. DHPG increased the frequency of sEPSCs without any change in their amplitude in the present study. This is inconsistent with the report by Fernandes et al. (2011) in which this agonist at the same concentration had no effects on sEPSCs in the rat NTS neurons. The discrepancy from our results remains unsolved, but may arise partly from the difference in species (guinea pig vs. rat) and recording neurons (second-order neuron vs. unidentified neuron). The DHPG-induced facilitation of sEPSC frequency was antagonized by LY367385 but not by MPEP. Furthermore, LY367385 by itself decreased the sEPSC frequency. These results

indicate that the mGluR1s are located at the axon terminals projecting from the intrinsic local network and play a facilitatory regulation in glutamatergic transmission. This is comparable with the findings that the activation of presynaptic group I mGluRs potentiates glutamate release in the rat cortex (Melendez et al., 2005; Moroni et al., 1998; Musante et al., 2008) and GABA release in the NTS (Jin et al., 2004). Our conclusion is supported by evidence obtained in condition where the extracellular concentration of glutamate was elevated by cystine or TBOA. Perfusion of cystine or TBOA increased the sEPSC frequency with the unaltered amplitude in second-order neurons. Cystine is an activator of cystine/glutamate antiporters that are expressed within



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Fig. 5 – (A) Antagonism of LY367385 or MPEP against TBOA-induced facilitation of sEPSCs in a second-order NTS neuron. Drugs were applied sequentially. Traces of sEPSCs taken before (control), during TBOA (100 μM) and co-perfusion of LY367385 (TBOAþLY367385 100 μM) or MPEP (TBOAþMPEP 10 μM). (B) Cumulative probability of the amplitude and inter-event interval (IEI) of sEPSCs for the neuron in (A). The distribution of IEI was significantly shifted to the leftward by TBOA (Po0.01 vs. control, Kolmogorov–Smirnov test), but that of the amplitude was unchanged (P40.05 vs. control, Kolmogorov–Smirnov test). (C) Relative values of the amplitude and frequency of sEPSCs. Values are the mean7SEM (n¼ 7). nPo0.05 vs. control (paired t-test). ♯Po0.05, N.S.; not significant (ANOVA followed by Bonferroni corrected multiple t-test). the NTS (Sato et al., 2002). TBOA is a competitive antagonist of glutamate transporters (EAAT1, 2) (Shimamoto et al., 1998). The concentration of TBOA (100 μM) or cystine (30 μM) used in the present study is estimated to elevate extracellular glutamate up to 3–4 μM according to the report by Oldenziel et al. (2007), although it is unclear that these agents work actually in the NTS similarly to those in the hippocampus (Oldenziel et al., 2007). Considering the EC50 value (at micromolar levels) of mGluRs (Hayashi et al., 1993), it is postulated that glutamate spillover from the synaptic clefts, which may cause extracellular glutamate buildup to enable mGluR activation, modulates glutamate release from the intrinsic local network neurons in a positive feedback manner. The increasing effect of sEPSC frequency by TBOA or cystine was completely

blocked by LY367385. Antagonism by the mGluR1 blocker is consistent with the results obtained in normal condition. These results suggest that facilitation of sEPSCs is derived from the presynaptic mGluR1 activation. Therefore, the mGluR1s may be functionally important for regulation of synaptic transmission by amplifying the network activity in the guinea pig NTS.

3.2. Modulation of NTS glutamatergic transmission by group II and III mGluRs Activation of the group II and III mGluRs by selective agonists decreased the amplitude of eEPSCs while actually increasing the PPR. Moreover, it decreased the frequency of sEPSCs


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Fig. 6 – Effects of mGluR agonists on eEPSCs in second-order NTS neurons. Traces of eEPSCs taken during before (control), 4 min after the onset of either DHPG (10 μM, A1), DCG IV (10 μM, B1) or L-AP4 (30 μM, C1), and 5 min after the washout (wash). Dashed lines indicate the control level of the first and second eEPSCs. (A2, B2, C2) Changes in the amplitude of first eEPSCs and the paired-pulse ratio. PPR was calculated as the peak amplitude of the second eEPSC divided by that of the first eEPSC. Data are the mean7SEM (n ¼5–6). nPo0.05, nnPo0.01, N.S.; not significant (ANOVA followed by Bonferroni corrected multiple t-test).

without effect on their amplitude. Our results are accordance with those obtained from the rat NTS neurons (Chen et al., 2002; Fernandes et al., 2011). Therefore, it is suggested that the group II and III mGluRs are located at the TS-afferent terminals and axon terminals of intrinsic interneurons and act as autoreceptors. Similar presynaptic inhibition by activation of the group II and/or III mGluRs has been reported in other areas including the spinal cord (Gerber et al., 2000) and the thalamus (Alexander and Godwin, 2005; Lam and Sherman, 2013). However, neither antagonist of the group II nor III mGluRs alone had any detectable effect on sEPSCs in the present study. Moreover, cystine or TBOA showed only excitatory effects on sEPSCs, and the sEPSC frequency did not decline to the lower level than control after co-perfusion of an mGluR1 antagonist with cystine or TBOA. Therefore it seems unlikely that the group II and III mGluRs work predominantly in the guinea pig NTS neurons. The reason for selective activation of the group I mGluRs (mGluR1s) was not clear. One conceivable possibility is the relatively closer location of mGluR1s to the synaptic cleft compared with that of the group II and III mGluRs. Since TBOA has been reported to induce elevation of glutamate concentration and subsequently activate both group I and III mGluRs in the hippocampal slices (Selkirk et al., 2003), ununiformity of

distribution of mGluRs is assumed in different central regions.

3.3.

Conclusions

Recently, it has been shown that synaptic activity generates glutamate spillover in the vicinity of active synapses (Okubo et al., 2010). Glutamate spillover is implicated in the activation of extrasynaptic glutamate receptors to regulate a variety of important neuronal and/or glial functions including synaptic transmission. However, the diverse effects of mGluR activation have been also reported in the different experimental conditions including animal species and recording areas. The heterogeneous activating properties of mGluRs seem to induce complicated modulation of synaptic transmission and physiological function (Browning and Travagli, 2007). In the present study, we demonstrated for the first time that the mGluR1s are present only on axon terminals of intrinsic glutamatergic neurons, and the group II and III mGluRs are expressed on axon terminals from intrinsic neurons and primary afferents in the guinea pig NTS neurons. Moreover, their contribution appears to act through presynaptic mechanisms as determined by the observations



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that the mGluR agonists or antagonists affected only the sEPSC frequency in the absence of a change in the amplitude and altered the eEPSC amplitude together with changes in the PPR. In conclusion, the present results lead to postulate that mGluR1s may be constitutively active and functionally important for homeostasis or reflex regulation by amplifying the network activity, whereas the group II and III mGluRs may not work under normal condition although their activation effectively dampens glutamatergic excitation.

4.

Experimental procedure

4.1.

Slice preparation

This study was approved by the Animal Care Committee at the Aichi Gakuin University and conducted in accordance with Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. Slice preparations were made as described previously (Haji and Ohi, 2010; Ohi et al., 2007, 2011). Briefly, male Hartley guinea pigs (200–400 g) were deeply anesthetized with inhalation of halothane and decapitated. The brainstem was excised and submerged in ice-cold low-calcium artificial cerebrospinal fluid (aCSF) containing (mM): NaCl, 125; KCl, 2.5; CaCl2, 0.1; MgCl2, 5; NaH2PO4, 1.25; D-glucose, 12.5; Lascorbic acid, 0.4; NaHCO3, 25. The pH was 7.4 when continuously bubbled with 95% O2-5% CO2. The brainstem was glued to the cutting stage of a vibrating slice cutter (Linear Slicer Pro 7, Dosaka, Kyoto, Japan) with the caudal side up. Two to three transverse slices of a 400-μm thickness including the NTS region, extending from 0.5 mm caudal to 1.2 mm rostral to the obex, were made, to which afferents of the superior laryngeal nerve project predominantly in guinea pigs (Ohi et al., 2005). The slices were incubated in standard aCSF (mM): NaCl, 125; KCl, 2.5; CaCl2, 2; MgCl2, 1.3; NaH2PO4, 1.25; D-glucose, 12.5; L-ascorbic acid, 0.4; NaHCO3, 25; saturated with 95% O2–5% CO2, for 30–40 min at 37 1C, and then kept at room temperature (2571 1C) until the recording. The slice was fixed in a recording chamber ( 0.4 ml volume, RC-26GLP, Warner Instruments, Hamden, CT, USA) under nylon mesh attached stainless anchor, then submerged in and continuously perfused with the standard aCSF at a flow rate of 1–2 ml/min. The neurons with small diameters (o15 μm) which may receive predominantly excitatory synaptic inputs (Champagnat et al., 1986; Kawai and Senba, 1996) were visually preselected in the medial and dorsal regions of NTS with an infrared-differential interference contrast videomicroscope (BX-51WI, Olympus, Tokyo; C2741, Hamamatsu Photonics, Hamamatsu, Japan).

4.2.

Whole-cell transmembrane current recording

Recordings were made at room temperature and under perfusion of aCSF including picrotoxin to block GABAA receptors in all experiments. The composition of the intracellular solution was (mM): potassium gluconate, 120; NaCl, 6; CaCl2, 5; MgCl2, 2; MgATP, 2; NaGTP, 0.3; EGTA, 10; HEPES, 10; pH 7.2 with KOH. The tip resistance of the electrodes ranged from 4 to 6 MΩ when filled with the pipette solution. After

9

establishing the cell-attached configuration with a seal resistance of 1–10 GΩ, the whole-cell mode was established with a brief negative current and pressure pulse. The series resistance (o30 MΩ) and membrane capacitance were compensated and checked regularly during the recording. At a holding potential of 60 mV, the transmembrane current was recorded with a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA, USA) with a high-cut filter at 2 kHz. The membrane current was sampled on-line at 4 kHz (PowerLab/4 s, AD Instruments, Castle Hill, Australia) and stored on hard disk of a computer. A stainless concentric bipolar electrode was placed on the TS ipsilateral to the recorded neuron. The distance between the two poles was 100 μm. The intensity of stimulation was set to a minimal voltage with which every pulse of the TS stimulation constantly induced a clear monosynaptic EPSC peak without failure. Usually, the stimulation intensity was 10–20 V and the pulse duration was 0.1 ms. Stimulation was given every 10 s. The EPSC with a short latency (o7.5 ms) and small jitter (SDo200 μs) from stimulus onset was judged to be monosynaptic (Champagnat et al., 1986; Doyle and Andresen, 2001). All neurons used in the present study were preselected as second-order neurons by these criteria. To investigate the site of action of mGluR agonists, PPR was calculated according to the method described previously (Debanne et al., 1996). For that, double stimulus pulses at an inter-pulse interval of 30 ms were applied to the TS to induce two serial eEPSCs.

4.3.

Drugs

The following agonists and antagonists of mGluRs were used and the concentrations of the former were determined at 2-fold EC50 and those of the latter at 10-fold IC50 to exert their action sufficiently, according to the previous reports (Chen et al., 2002; Fernandes et al., 2011; Kingston et al., 1998, 2002; Morishita and Alger, 2000): DHPG ((RS)-3,5-dihydroxyphenylglycine; 10 μM, an agonist of group I mGluRs, Tocris, Ellisville, Missouri, USA), LY367385 ((S)-(þ)-α-amino-4-carboxy-2methylbenzeneacetic acid; 100 μM, an antagonist of mGluR1s, Tocris), MPEP (2-methyl-6-(phenylethynyl) pyridine; 10 μM, an antagonist of mGluR5s, Wako, Osaka, Japan), DCG IV glycine; 10 μM, ((2S,20 R,30 R)-2-(20 ,30 -dicarboxycyclopropyl) an agonist of group II mGluRs, Tocris), LY341495 ((2S)-2Amino-2-[(1S,2S)-2-carboxycy cloprop-1-yl]-3-(xanth-9-yl) propanoic acid; 1 μM, an antagonist of group II mGluRs, Tocris), L-AP4 (L-(þ)-2-amino-4-phosphonobutyric acid; 30 μM, an agonist of group III mGluRs, Tocris), CPPG ((RS)-αCyclopropyl-4-phosphonophenyl glycine; 200 μM, an antagonist of group III mGluRs, Tocris). Referring to the previous report (Oldenziel et al., 2007), cystine (30 μM, an activator of cystine/glutamate exchanger, Wako) and TBOA (DL-threo-βbenzyloxyaspartate; 100 μM, an inhibitor of EAAT, Tocris) were used to elevate extracellular glutamate. CNQX (6-cyano-7-nitroquinoxaline-2, 3-dione disodium; 10 μM, Sigma, St Louis, MO, USA) and picrotoxin (100 μM, Sigma) were also used (Ohi et al., 2007). All drugs were dissolved in aCSF and applied for 5 min by gravity feed from 60 ml reservoirs bubbled with 95% O2–5% CO2. It took about 60 s to compensate for dead space of tubing between the bath and reservoirs.


BRES : 43532

10

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4.4.


Data acquisition and analysis

The recorded membrane currents were analyzed off-line with Chart 5 and Scope 4 (AD Instruments) and ORIGIN software (Origin Lab, Northampton, MA, USA). The threshold for detection of sEPSCs was set just above baseline noises of the recordings, which was 5–10 pA. The number and amplitude of sEPSCs occurring for 1 min were calculated. Averaged traces of eEPSCs were made by adding 5–10 sampled data using stimulus pulses as a trigger. The amplitude of eEPSC was calculated as the difference between the post-stimulus peak current and the pre-stimulus mean current over 10 ms. PPR was defined as the peak amplitude of the second eEPSC divided by that of the first eEPSC. For the group analysis, the data were compared before (control), during drug application (4 min after the onset of drug perfusion) and during washout (5 min after the onset of washout). Group values are expressed as the mean7SEM. Derived parameters were compared using a one-way analysis of variance (ANOVA) followed by multiple comparisons (Bonferroni corrected multiple t-test), paired t-test or Kolmogorov–Smirnov test with the significant level set at Po0.05.

Disclosures No conflicts of interest, financial or otherwise, are declared by the authors.

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Influence of nucleus tractus solitarius stimulation and baroreceptor activation on rat parabrachial neurons.

Arterial chemoreceptor input to nucleus tractus solitarius.

GABA in nucleus tractus solitarius participates in electroacupuncture modulation of cardiopulmonary bradycardia reflex.

Glutamate receptors in the nucleus tractus solitarius contribute to ventilatory acclimatization to hypoxia in rat.

Neurons in the nucleus tractus solitarius mediate the acupuncture analgesia in visceral pain rats.

An Essential Role of Fyn in the Modulation of Metabotropic Glutamate Receptor 1 in Neurons.

Electrical stimulation of nucleus tractus solitarius excites vagal preganglionic cardiomotor neurons of the nucleus ambiguus in rats.

Projections from the nucleus and tractus solitarius in the cat.

Activation of metabotropic glutamate receptors regulates ribosomes of cochlear nucleus neurons.

An atlas of the rat subpostremal nucleus tractus solitarius.

Modulation of brown adipose tissue-mediated thermogenesis by lesions to the nucleus tractus solitarius in the rat.

Cardiorespiratory response patterns elicited by microinjections of neuropeptide Y in the nucleus tractus solitarius.

Acute intermittent optogenetic stimulation of nucleus tractus solitarius neurons induces sympathetic long-term facilitation.

[The nucleus tractus solitarius: neuroanatomic, neurochemical and functional aspects].

Developmental decline in modulation of glutamatergic synapses in layer IV of the barrel cortex by group II metabotropic glutamate receptors.

Excitatory amino acids may mediate nucleus tractus solitarius input to rat parabrachial neurons.

Direct modulation of GABAA receptor by intracellular ATP in dissociated nucleus tractus solitarii neurones of rat.

Polysaccharides as taste stimuli: their effect in the nucleus tractus solitarius of the rat.

Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation.

Mechanisms Associated with Activation of Intracellular Metabotropic Glutamate Receptor, mGluR5.

Presynaptic modulation of glutamate and dynorphin release by excitatory amino acids in the guinea-pig hippocampus.

Responses of Nucleus Tractus Solitarius (NTS) early and late neurons to blood pressure changes in anesthetized F344 rats.

Localization and retention in vitro of fluorescently labeled aortic baroreceptor terminals on neurons from the nucleus tractus solitarius.

Endogenous GLP-1 acts on paraventricular nucleus to suppress feeding: projection from nucleus tractus solitarius and activation of corticotropin-releasing hormone, nesfatin-1 and oxytocin neurons.