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Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. Feb 1, 2002; 538(Pt 3): 773–786.
PMCID: PMC2290096

Synaptic transmission in nucleus tractus solitarius is depressed by Group II and III but not Group I presynaptic metabotropic glutamate receptors in rats

Abstract

Presynaptic metabotropic glutamate receptors (mGluRs) serve as autoreceptors throughout the CNS to inhibit glutamate release and depress glutamatergic transmission. Both presynaptic and postsynaptic mGluRs have been implicated in shaping autonomic signal transmission in the nucleus tractus solitarius (NTS). We sought to test the hypothesis that activation of presynaptic mGluRs depresses neurotransmission between primary autonomic afferent fibres and second-order NTS neurones. In second-order NTS neurones, excitatory postsynaptic currents (EPSCs) synaptically evoked by stimulation of primary sensory afferent fibres in the tractus solitarius (ts) and currents postsynaptically evoked by α-amino-3-hydroxy-4-isoxazoleproprionic acid (AMPA) were studied in the presence and absence of mGluR agonists and antagonists. Real-time quantitative RT-PCR (reverse transcription-polymerase chain reaction) was used to determine whether the genes for the mGluR subtypes were expressed in the cell bodies of the primary autonomic afferent fibres. Agonist activation of Group II and III but not Group I mGluRs reduced the peak amplitude of synaptically (ts) evoked EPSCs in a concentration-dependent manner while having no effect on postsynaptically (AMPA) evoked currents recorded in the same neurones. At the highest concentrations, the Group II agonist, (2S,3S,4S)-CCG/(2S,1′S,2′S)-2-carboxycyclopropyl (l-CCG-I), decreased the amplitude of the ts-evoked EPSCs by 39 % with an EC50 of 21 μm, and the Group III agonist, l(+)-2-amino-4-phosphonobutyric acid (l-AP4), decreased the evoked EPSCs by 71 % with an EC50 of 1 mm. mRNA for all eight mGluR subtypes was detected in the autonomic afferent fibre cell bodies in the nodose and jugular ganglia. Group II and III antagonists ((2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG) and (RS)-α-methylserine-O-phosphate (MSOP)), at concentrations that blocked the respective agonist-induced synaptic depression, attenuated the frequency-dependent synaptic depression associated with increasing frequencies of ts stimulation by 13–34 % and 13–19 %, respectively (P < 0.05, for each). We conclude that Group II and III mGluRs (synthesized in the cell bodies of the primary autonomic afferent fibres and transported to the central terminals in the NTS) contribute to the depression of autonomic signal transmission by attenuating presynaptic release of glutamate.

Synapses in the nucleus tractus solitarius (NTS) are the first regulatory moments in the CNS for modulating autonomic signal transmission and hence reflex function (Loewy & McKellar, 1980; Loewy, 1990; Spyer, 1990). The autonomic signals are transmitted from the first-order fibres to second-order NTS neurones by glutamate acting at ionotropic glutamate receptors (Andresen & Yang, 1990; Aylwin et al. 1997). One potential mechanism for modulating the fast glutamatergic transmission is the coincident activation of G-protein-coupled metabotropic glutamate receptors (mGluRs) (Burke & Hablitz, 1994; Crépel et al. 1994; Tainnie et al. 1994; Manzoni et al. 1995; Bushell et al. 1996; Conn & Pin, 1997; Scanziani et al. 1997; Schrader & Tasker, 1997; Hay et al. 1999; Cartmell & Schoepp, 2000). Throughout the CNS, glutamatergic transmission has been shown to be either augmented or reduced depending on the specific mGluR subtypes present at the synapses, their prevalence at presynaptic versus postsynaptic sites, and the frequency of afferent input. In general, Group I mGluRs (subtypes 1 and 5) are predominantly located on the cell soma, activate phospholipase C, and increase neuronal excitability (Pin & Duvoisin, 1995; Conn & Pin, 1997). Group II (subtypes 2 and 3) and Group III (subtypes 4, 6, 7 and 8) receptors are located predominantly on presynaptic terminals, are negatively coupled to adenylyl cyclase, and are autoreceptors, inhibiting glutamate release to reduce synaptic transmission (Scanziani et al. 1997; Cartmell & Schoepp, 2000).

One prominent feature of autonomic signal transmission in the NTS is a frequency-dependent synaptic depression associated with a relative reduction in the amount of glutamate released from the presynaptic terminal with increasing frequencies of afferent input (Miles, 1986; Felder & Heesch, 1987; Mifflin & Felder, 1988; Schild et al. 1995). We and others have provided evidence that this frequency-dependent depression is, at least in part, presynaptically mediated (Felder & Heesch, 1987; Mifflin & Felder, 1988; Schild et al. 1995; Chen et al. 1999).

We proposed that one presynaptic mechanism might be activation of the presynaptic mGluRs. Although presynaptic mechanisms cannot be resolved with certainty with extracellular recordings of action potential firing, we provided supportive evidence by showing that, in the whole animal, agonist activation of Group II mGluRs depressed synaptic transmission at NTS baroreceptor synapses, and that blockade of the Group II receptors attenuated the depression of synaptic transmission at high input frequencies (Liu et al. 1998).

The purpose of the present study was to more directly test the hypothesis that presynaptic mGluR activation reduces autonomic signal transmission between primary afferent fibres and second-order NTS neurones. If the hypothesis is true, then: (1) mGluR agonists should decrease the amplitude of glutamatergic EPSCs synaptically evoked by low-frequency stimulation of the tractus solitarius (ts) while having no inhibitory effect on postsynaptically evoked currents in the same neurones by exogenous application of the ionotropic glutamate receptor agonist, α-amino-3-hydroxy-4-isoxazoleproprionic acid (AMPA); (2) mRNAs encoding the mGluR subtypes must be synthesized in the cell bodies (nodose and jugular ganglia) of the primary afferent fibres (an essential step if the receptors are to be moved along their central axons and inserted at the terminals); and (3) mGluR antagonists should attenuate the frequency-dependent presynaptic depression.

METHODS

All experimental protocols in this work were reviewed and approved by the Institutional Animal Care and Use Committee in compliance with the Animal Welfare Act and in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Slice preparation for whole-cell recordings

Male Sprague-Dawley rats, 3–4 weeks old, were anaesthetized with a combination of ketamine (35 mg kg−1) and xylazine (2 mg kg−1) and decapitated. The brain was rapidly exposed and submerged in ice-cold (< 4 °C) high-sucrose artificial cerebrospinal fluid (aCSF) that contained (mm): 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 220 sucrose and 2 CaCl2; the pH was 7.4 when continuously bubbled with 95 % O2/5 % CO2 (300 mosmol kg−1). Brainstem coronal slices (250 μm thick) were cut with the Vibratome 1000 (Technical Products International, St Louis, MO, USA). After incubation for 45 min at 37 °C in high-sucrose aCSF, the slices were placed in normal aCSF that contained (mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose and 2 CaCl2; the pH was 7.4 when continuously bubbled with 95 % O2/5 % CO2 (300 mosmol kg−1). During the experiments, a single slice was transferred to the recording chamber, held in place with a nylon mesh, and continuously perfused with oxygenated aCSF at a rate of approximately 3 ml min−1. All experiments were performed at 33–34 °C.

Whole-cell voltage-clamp recording

Borosilicate glass electrodes were filled with a CsF solution containing (mm): 145 CsF, 5 NaCl, 1 MgCl2, 3 K-ATP, 0.2 Na-GTP, 10 EGTA and 10 Hepes; pH 7.4 (300 mosmol kg−1). Whole-cell voltage-clamp recordings were made in NTS neurones with the Axoclamp 1D patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). Whole-cell currents were filtered at 2 kHz, digitized at 10 kHz with the DigiData 1200 Interface (Axon Instruments) and stored in a 386 DX computer. Data were analysed off-line using the pCLAMP6 software (Axon Instruments). The seal resistance was > 1 GΩ, and the series resistance was < 25 MΩ.

Neurones included in this study were voltage clamped at −60 mV and met the following presumptive criteria for being classified as second order: (1) an EPSC was consistently evoked by each of two ts stimuli separated by 5 ms (200 Hz) and (2) the variability of the onset latency of the evoked EPSCs was ≤ 0.5 ms (Miles, 1986; Scheuer et al. 1996). Stimulating voltages (1–10 V, 0.1 ms square wave pulses) were delivered through bipolar tungsten electrodes (1 μm tips separated by 80 μm) to the ts ipsilateral to the recording site. To eliminate the potential influence of activation of inhibitory interneurones, ts-evoked EPSCs were pharmacologically isolated by constant perfusion with the specific GABAA receptor antagonist, bicuculline (10 μm).

Protocols

Protocol 1 was designed to examine the extent to which mGluR agonists decrease the peak amplitude of EPSCs monosynaptically evoked by low-frequency stimulation of fibres in the ts. Following classification of the neurone as second order, the ts was continuously stimulated at 0.1–0.2 Hz and evoked EPSCs were recorded for 1 min during the control period and for 3 min during perfusion with one of the following selective mGluR agonists: (S)-3,5-dihydroxyphenylglycine (3,5-DHPG, Group I, 10 μm-3 mm), (2S,3S,4S)-CCG/(2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (l-CCG-I, Group II, 3 μm-300 μm), or l(+)-2-amino-4-phosphonobutyric acid (l-AP4, Group III, 3 μm-3 mm). The concentrations for each agonist were applied in a random order, and evoked EPSCs were recorded for 3 min during perfusion with each concentration. Since the Group I agonist, 3,5-DHPG, did not depress the synaptic transmission at the highest concentration used (3 mm), no further experiments with Group I were performed.

Protocol 2 was implemented to further determine whether the mGluR agonists acted at presynaptic sites to depress synaptic transmission by comparing the peak amplitude of EPSCs synaptically evoked by ts stimulation and currents postsynaptically evoked by AMPA (0.5 mm, 20–50 μl) in the same neurone in the absence and presence of l-CCG-I (20 μm) or l-AP4 (1 mm). AMPA was injected with a 0.5 ml insulin syringe that was connected by polyethylene-10 tubing to an inlet of the main perfusion line. Tractus solitarius-evoked EPSCs were established by averaging the peak EPSC amplitudes evoked by five consecutive ts stimuli delivered at 0.2 Hz. Since AMPA injected into the perfusion line could potentially activate neurones synapsing onto the voltage-clamped NTS neurone, all action potentials in the NTS network were blocked with tetrodotoxin to isolate the measured AMPA-evoked current to those AMPA receptors activated on the voltage-clamped neurone. Specifically, following classification of the neurone as second order, tetrodotoxin was added to the perfusion (1 μm final concentration). Synaptic blockade was confirmed by the complete blockade of ts-evoked EPSCs, then the peak amplitudes of the AMPA-evoked currents were compared in the absence and presence of l-CCG-I (20 μm) or l-AP4 (1 mm).

For the mGluRs to be inserted into the presynaptic terminals of the primary afferent fibres to modulate synaptic transmission, the mRNA must be copied from the gene that encodes the protein. Protocol 3 used real-time RT-PCR (reverse transcription- polymerase chain reaction; see below) to document whether gene expression for each mGluR subtype could be detected in the cell bodies of the primary afferent fibres, located in the nodose or jugular ganglia.

The rationale for protocol 4 was two-fold: First, in order to test the effect of the mGluR antagonists on frequency-dependent depression, we established the appropriate concentration of each antagonist. Specifically, we determined the appropriate concentration of the Group II mGluR antagonist required to block the effect of the Group II agonist and the appropriate concentration of the Group III antagonist required to block the effect of the Group III agonist. Second, although the antagonists are fairly selective, we verified their selectivity at the concentrations used in this study by showing that the Group II receptor antagonist did not block the effect of the Group III agonist and that the Group III receptor antagonist did not block the effect of the Group II agonist. As in protocol 1, following classification of the neurone as second order, the ts was continuously stimulated at low frequencies of 0.1–0.2 Hz. Evoked EPSCs were recorded for 1 min during the control period and for 3 min during perfusion with either l-CCG-I (Group II, 30 μm or 100 μm) or l-AP4 (Group III, 1 mm or 3 mm) for 3 min. After a washout period (3 min), the slice was perfused with the selective Group II antagonist, (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG, 200 μm), to block the effect of l-CCG-I, or with the selective Group III antagonist, (RS)- α-methylserine-O-phosphate (MSOP, 3 mm), to block the l-AP4 effect. The antagonist was applied for 6 min; during the last 3 min, the agonist was added to the perfusate. In separate experiments, the Group II antagonist (MCCG, 200 μm) was tested against the Group III agonist (l-AP4, 1 mm) and the Group III antagonist (MSOP, 3 mm) against the Group II agonist (l-CCG-I, 30 μm).

Protocol 5 was designed to examine the extent to which the mGluR antagonists attenuated the frequency-dependent synaptic depression. Trains of 30 ts stimuli were delivered at frequencies of 0.4, 3, 9, 24 or 48 Hz in randomized order. A control EPSC was established by averaging the peak EPSC amplitudes evoked by five consecutive ts stimuli delivered at 0.2 Hz before each train of stimuli at every frequency. The frequency-dependent depression was determined in the absence and presence of one of the mGluR antagonists (starting at 3 min into the antagonist perfusion): MCCG (200 μm) or MSOP (3 mm). To determine the magnitude of synaptic depression, peak EPSC amplitudes were averaged over the last 10 of the 30 stimuli at every frequency and expressed as a percentage of the control EPSC. In pilot studies (n = 7), the frequency-dependent depression was determined twice in aCSF (same time interval as used in antagonist tests) and the depression was not different between the two trials (ANOVA: P < 0.0001, frequency; P = 0.3975, trials; P = 0.2135, interaction). The synaptic depression at 0.4, 3, 9, 24 and 48 Hz was 85 ± 3 %, 60 ± 5 %, 40 ± 7 %, 18 ± 5 % and 11 ± 4 % for the first trial and 80 ± 3 %, 57 ± 6 %, 37 ± 7 %, 21 ± 6 % and 13 ± 4 % for the second trial, respectively (means ± s.e.m.).

Real-time quantitative RT-PCR

Male Sprague-Dawley rats, 3–4 weeks old, were anaesthetized with a combination of ketamine (35 mg kg−1) and xylazine (2 mg kg−1). After decapitation, the nodose and jugular ganglia were isolated and stored in 1.5 ml microcentrifuge tubes containing 75 % ethanol at 4 °C until the total RNA was isolated.

Total RNA was isolated from both nodose and both jugular ganglia (n = 10 animals) using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA) and quantified by spectrophotometry at 260 nm. Gene expression analysis was performed using real-time RT-PCR (GeneAmp 5700 Sequence Detection System, PE Biosystem, Foster City, CA, USA). For the reverse transcription, 30 ng of total RNA was treated with DNase I and then used to synthesize cDNA by the ThermoScript RT-PCR System (Invitrogen Co., Carlsbad, CA, USA). Random hexamers were used for the reverse transcription. PCR primers were designed using PrimerExpress software (PE Biosystem) and are shown in Table 1. One microlitre of each cDNA mixture was used for the PCR for each mGluR gene. The standard for each gene was synthesized from the cDNAs through PCR consisting of 1 × PCR buffer II (PE Biosystem); 3 mm MgCl2; 200 μm each of dATP, dCTP, dGTP and dTTP; 300 nm of appropriate primers and 1.25 U of AmpliTaq Gold DNA Polymerase (PE Biosystem). PCR was carried out using the following thermal cycling parameters: 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. Amplified DNA fragments were gel purified using QIAEX II Gel Extraction Kit (Qiagen Inc.), then quantified by spectrophotometry at 260 nm. A ‘house-keeping’ gene, GAPDH, was used as an endogenous control to account for variability in the conversion efficiency of the reverse transcription reaction. The PCR fragment of each gene was sequence confirmed (data not shown). Water was used as a negative control for false-positive results.

Table 1
Primer accession numbers, sequences and positions in the open reading frame

For quantification, standard curves were determined from serial dilutions (10−6, 10−7, 10−8 and 10−10) of known amounts of each purified gene fragment of the target genes and GAPDH. Each sample and each selected dilution was amplified in quadruplicate using real-time quantitative PCR. The thermocycling parameters were: 50 °C for 2 min, 95 °C for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The constituents were 1× SYBR Green PCR Buffer (PE Biosystem); 3 mm MgCl2; 200 μm each of dATP, dCTP and dGTP; 400 μm dUTP; 300 nm of each primer; 0.5 U of AmpErase Uracil N-glycosylase; and 1.25 U of AmpliTaq Gold DNA Polymerase. For all experimental samples, the values for the target genes (mGluR subtypes 1–8) were expressed as copy number per nanogram of total RNA and also normalized to GAPDH.

Data analysis

Data are expressed as means ± s.e.m. unless otherwise indicated. Differences were considered significant at P < 0.05. The onset latency for each cell was determined from 10 evoked EPSCs delivered at 0.2 Hz. To determine the variability of the onset latency, we determined the range (the difference between the longest and the shortest onset latency) and the variance (the dispersion of the data about the mean onset); both were calculated from the 10 EPSCs.

For protocol 1, to determine the effect of the mGluR agonist on synaptic transmission, the average peak amplitude of the EPSCs, evoked by ts stimulation (0.1–0.2 Hz) during the last minute of agonist perfusion, was expressed as a percentage of the average peak amplitude of the EPSCs evoked during the last minute of the control period before agonist perfusion. The agonist concentration-response curve for each group was analysed using a one-way ANOVA. The agonist concentration-response curves were fitted to a logistic function to obtain the EC50 and the concentration range for 10–90 % of the maximum depression.

For protocol 2, to compare the effect of agonist on synaptically and postsynaptically evoked currents, the peak amplitude of the EPSCs synaptically evoked by ts stimulation and currents postsynaptically evoked by AMPA were compared in the presence and absence of the agonist with a paired t test.

For protocol 4, to determine the concentration of antagonist that blocked the agonist effect, the average peak amplitude of EPSCs evoked in the last minute of agonist perfusion was expressed as a percentage of the average peak amplitude of the EPSCs evoked during the control period. During the perfusion of the antagonist, the average peak amplitude of the EPSCs evoked during the respective agonist perfusion was expressed as a percentage of the average peak amplitude of the EPSCs evoked during antagonist perfusion before agonist. The effect of antagonist on the same group agonist-induced reduction in peak EPSC amplitude was compared using a paired t test. A paired t test was used to analyse the effect of Group III antagonist on Group II agonist and the effect of Group II antagonist on Group III agonist.

For protocol 5, the effect of antagonist on frequency-dependent depression was analysed using a two-way ANOVA with the absence and presence of antagonist as one within factor and the frequency as the other within factor.

Drugs

The mGluR agonists (3,5-DHPG, l-CCG-I and l-AP4) and antagonists (MCCG and MSOP) were obtained from Tocris (Ballwin, MO, USA). Bicuculline was obtained from RBI (Natick, MA, USA). CsF, EGTA and Hepes were obtained from Sigma (St. Louis, MO, USA). All other chemicals were obtained from Fisher (Pittsburgh, PA, USA).

RESULTS

All electrophysiological data were obtained from intermediate and caudal NTS neurones in which EPSCs met the presumptive criteria for monosynaptic activation, responding to each of the two stimuli separated by 5 ms and having a short onset latency (1.87 ± 0.55 ms; mean ± s.d.) with a small range (0.20 ± 0.13 ms; mean ± s.d.) and variance (0.011 ± 0.009 ms; mean ± s.d.). The peak amplitude of the ts-evoked EPSCs in the control conditions across all protocols averaged 214 ± 86 pA (mean ± s.d.) and ranged from 49 to 448 pA.

Effect of mGluR agonists on tractus solitarius (ts)-evoked EPSCs

The Group II agonist, l-CCG-I, depressed the synaptic transmission between the primary afferent fibres and presumed second-order NTS neurones in a concentration-dependent manner (Fig. 1). The traces in Fig. 1A show the depressant effect of l-CCG-I (10, 30 and 300 μm) on the peak amplitude ts-evoked EPSCs in one neurone: the EPSC peak amplitude was decreased from 171 pA in the control condition to 159 pA (93 % of control) with 10 μm l-CCG-I; from 169 to 135 pA (80 % of control) with 30 μm l-CCG-I; and from 182 to 115 pA (63 % of control) with 300 μm l-CCG-I. Figure 1B shows the peak amplitudes of successively evoked EPSCs before, during and after each concentration of l-CCG-I. The group data (Fig. 1C), fitted to a sigmoid function, illustrates the concentration-dependent synaptic depression (one-way ANOVA: P = 0.002). The maximum effect of l-CCG-I was a decrease in the peak amplitude of the evoked EPSC to 61 % of the control value. Based on the curve fit result, the EC50 was 21 μm, and the concentration range for 10–90 % for the maximum depression was 11–44 μm.

Figure 1
The Group II agonist, l-CCG-I, depressed synaptic transmission in NTS neurones in a concentration-dependent manner

The Group III mGluR agonist, l-AP4, also depressed synaptic transmission in a concentration-dependent manner (Fig. 2). The traces in Fig. 2A show the effect of l-AP4 (100, 300 and 1000 μm) on the peak amplitude ts-evoked EPSCs in one neurone: the two higher concentrations of l-AP4 (300 and 1000 μm) depressed the EPSC amplitudes from a control of 319 pA to 278 pA (87 % of the control) and 304 pA to 204 pA (67 % of the control), respectively. Figure 2B shows the peak amplitudes of successively evoked EPSCs before, during and after each concentration of l-AP4. The group data (Fig. 2C), fitted to a sigmoid function, confirmed the concentration-dependent synaptic depression (one-way ANOVA: P < 0.005). Given that the concentration-response curve had no plateau for the maximum inhibition, we set the value for the asymptotic minimum at zero for the curve fit. From the curve fit results, we estimated the EC50 as 1 mm and the concentration range for 10–90 % for the maximum depression as 55 μm to 22 mm.

Figure 2
The Group III agonist, l-AP4, depressed synaptic transmission in NTS neurones in a concentration-dependent manner

The Group I mGluR agonist, 3,5-DHPG, had no effect on synaptic transmission in the NTS at any concentration tested (109 ± 8 %, 97 ± 6 %, 97 ± 19 %, 97 ± 10 %, 87 ± 2 % and 94 ± 10 % for 10, 30, 100, 300, 1000 and 3000 μm respectively, one-way ANOVA: P = 0.767, n = 3–6).

Evidence for presynaptic sites of l-CCG-I and l-AP4 effects

To provide evidence as to whether the Group II and III agonists depressed synaptic transmission by a presynaptic mechanism, we determined the effects of the respective EC50 of l-CCG-I and l-AP4 on synaptically (ts) evoked EPSCs and postsynaptically (AMPA) evoked currents. As shown in the example (Fig. 3A, top traces), l-CCG-I (20 μm) depressed the peak amplitude of ts-evoked EPSCs (from 139 pA to 108 pA, a decrease of 22 %) in the same neurone in which the agonist had no effect on the AMPA-evoked current (Fig. 3A, bottom traces); the peak amplitude of the AMPA-evoked current was 120 pA before and 127 pA during l-CCG-I. The group data (Fig. 3B, n = 6) confirm that l-CCG-I significantly depressed the peak amplitude of synaptically (ts) evoked EPSCs (paired t test: P = 0.016), while having no effect on postsynaptically (AMPA) evoked currents in the same neurones (paired t test: P = 0.2). We further confirmed that l-CCG-I also had no effect on the peak amplitude of AMPA-evoked currents when synaptic transmission was blocked: as shown in Fig. 3C, l-CCG-I (20 μm) did not affect the AMPA-evoked currents in the presence of tetrodotoxin (n = 6, paired t test: P = 0.42).

Figure 3
Tractus solitarius (ts)-evoked EPSCs and AMPA-evoked currents under control conditions (aCSF) and in the presence of Group II agonist, l-CCG-I (20 μm)

The Group III agonist, l-AP4 (1 mm), markedly depressed the peak amplitude of synaptically (ts) evoked EPSCs while having no consistent effect on the postsynaptically (AMPA) evoked currents. As shown in the example (Fig. 4A, top traces), l-AP4 depressed the peak amplitude of the ts-evoked EPSCs (from 292 pA to 143 pA, a decrease of 49 %) in the same neurone in which the AMPA-evoked current was actually enhanced from 106 pA to 171 pA (Fig. 4A, bottom traces). As shown in the group data (Fig. 4B, n = 9), while l-AP4 significantly depressed the synaptically evoked EPSCs (paired t test: P = 0.001), the agonist had no consistent effect on the postsynaptically evoked currents in the same neurones (paired t test: P = 0.99). As shown in Fig. 4C, l-AP4 (1 mm) also had no consistent effect on the AMPA-evoked currents when synaptic transmission was blocked with tetrodotoxin (n = 6, paired t test: P = 0.90).

Figure 4
Tractus solitarius (ts)- and AMPA-evoked currents in the same neurone under control conditions (aCSF) and in the presence of Group III agonist, l-AP4 (1 mm)

To determine whether the mGluRs were synthesized in the autonomic afferent fibres, we examined the gene expression in the cell bodies in the nodose and jugular ganglia. Figure 5A shows an example of the real-time PCR reaction of the standard curve (with serial dilutions) and one sample for mGluR subtype 7. The fluorescence level of SYBR Green I dye (Rn) was plotted against the PCR reaction cycle number. The threshold cycle (CT) at which the PCR product was first detected (the horizontal dotted line) was determined for each standard dilution and sample. The copy number of the mRNA in the reaction is linearly related to CT and was used to calculate the copy number of the mRNA in the sample (Fig. 5B). The mRNAs for all eight mGluR subtypes were expressed (Fig. 6; n = 10 animals). The group data were expressed as copy number per nanogram of total RNA and were also normalized to a house-keeping gene, GAPDH, to account for variability in the conversion efficiency of the reverse transcription reaction. The relative gene expression level between the eight subtypes of the mGluRs was the same whether the data were expressed as absolute copy number (Fig. 6, top panels) or normalized to GAPDH (Fig. 6, bottom panels).

Figure 5
An example of amplification of the mGluR 7 gene of serial dilutions of mGluR 7 standard, from 5.95 × 104 to 59.5 copies, and one sample in four replicates
Figure 6
mRNA from all mGluR subtypes was present in the nodose and jugular ganglia

Antagonist blockade of agonist effects

l-CCG-I depression of the synaptically evoked EPSCs was prevented by the selective Group II antagonist, MCCG (200 μm), as shown in the example (Fig. 7A and B) and by the group data (Fig. 7C). Shown in the example (Fig. 7A, left traces), l-CCG-I (100 μm) reduced the peak amplitude of the ts-evoked EPSCs in one neurone from 197 pA in the control condition to 132 pA (33 % decrease). MCCG (200 μm) prevented the l-CCG-I-induced depression (Fig. 7A, right traces); the ts-evoked EPSC was 163 pA under control conditions and 160 pA in the presence of l-CCG-I plus MCCG. Figure 7B shows the peak amplitude of successively evoked EPSCs before, during and after l-CCG-I and then before, during and after MCCG plus l-CCG-I. In the presence of MCCG, the same concentration of l-CCG-I had no effect on the EPSCs. The group data (Fig. 7C) illustrate the MCCG attenuation of the synaptic depression induced by l-CCG-I 30 μm (n = 5) and 100 μm (n = 4) (paired t test: P = 0.03 and 0.04, respectively). l-CCG-I (30 and 100 μm, respectively) resulted in 30 ± 4 % and 39 ± 4 % decreases in the amplitudes of the ts-evoked EPSCs; the decreases were blocked by the antagonist, MCCG. While MCCG attenuated the LCCG-I inhibitory effect, the antagonist had no effect on the EPSCs evoked by stimulation of the ts at these low frequencies (0.1–0.2 Hz) under control conditions (data not shown).

Figure 7
l-CCG-I-induced synaptic depression was blocked by the selective Group II antagonist, MCCG

The l-AP4-evoked synaptic depression was blocked by the selective Group III antagonist, MSOP, as shown in the example (Fig. 8A and B) and group data (Fig. 8C). In the example (Fig. 8A, left traces), l-AP4 (3 mm) reduced the peak amplitude of the ts-evokedEPSCs in one neurone from 243 pA in the control condition to 36 pA (85 % decrease). MSOP (3 mm) prevented the l-AP4-induced depression (Fig. 8A, right traces); the ts-evoked EPSC was 217 pA under control conditions and 166 pA in the presence of l-AP4 plus MSOP. Figure 8B shows the peak amplitude of successively evoked EPSCs before, during and after l-AP4, and then before, during and after MSOP plus l-AP4. In the presence of MSOP, the same amount of l-AP4 had no effect on the peak amplitude of the evoked EPSCs. The group data (Fig. 8C) confirmed the MSOP-evoked attenuation of the effects of 1 mm (n = 6) and 3 mm (n = 6) of l-AP4 (paired t test: P = 0.02 and 0.01, respectively). l-AP4 (1 and 3 mm, respectively) resulted in 36 ± 6 % and 76 ± 7 % decreases in the amplitudes of the ts-evoked EPSCs; the decreases were prevented by the antagonist, MSOP. The antagonist had no effect on the EPSCs evoked by this low frequency (0.1–0.2 Hz) stimulation of the ts under control conditions (data not shown).

Figure 8
l-AP4-induced synaptic depression was blocked by the selective Group III antagonist, MSOP

We performed additional experiments to confirm the selectivity of l-AP4 for Group III receptors and of l-CCG-I for Group II receptors in mediating the synaptic depression. A concentration close to the EC50 was selected for the agonists (1 mm for l-AP4 and 30 μm for l-CCG-I). As shown in the group data (Fig. 9), the l-AP4-induced depression of the peak amplitude of the ts-evoked EPSCs in aCSF was not changed in the presence of the Group II antagonist, MCCG, at the concentration (200 μm) that blocked the l-CCG-I-induced synaptic depression (n = 6, paired t test: P = 0.8). Similarly, the l-CCG-I-induced depression of the peak amplitude of the ts-evoked EPSCs in aCSF was not changed in the presence of the Group III antagonist, MSOP, at the concentration (3 mm) that blocked the l-AP4-induced synaptic depression (n = 6, paired t test: P = 0.6).

Figure 9
Group data showing the effect of the Group II antagonist (MCCG) on the Group III agonist (l-AP4)-induced synaptic depression, and the effect of the Group III antagonist (MSOP) on the Group II agonist (l-CCG-I)-induced synaptic depression

Effect of mGluRs on frequency-dependent synaptic depression

Since Group II and III agonists depressed low-frequency synaptic transmission, we tested the respective antagonists on the synaptic depression associated with high-frequency afferent input. The Group II antagonist, MCCG (200 μm), attenuated the frequency-dependent synaptic depression (Fig. 10). The example shown in Fig. 10A illustrates the synaptic depression at ts stimulation frequencies of 3, 9 and 24 Hz in the absence and presence of MCCG. In each trace, the dotted line represents the averaged peak amplitude of five EPSCs evoked by ts stimuli delivered at 0.2 Hz, and the continuous line represents the averaged peak amplitude of the last 10 EPSCs evoked by 30 ts stimuli delivered at 3, 9 and 24 Hz. In the absence of MCCG (aCSF), the peak amplitudes of the EPSCs decreased in a frequency-dependent manner. MCCG attenuated the synaptic depression at each frequency. The group data are shown in Fig. 10B. MCCG, at a concentration that blocked the l-CCG-I-induced synaptic depression and spared the l-AP4-induced synaptic depression, significantly attenuated the frequency-dependent depression (two-way ANOVA: P = 0.049, MCCG; P < 0.001, frequency; P = 0.043, interaction).

Figure 10
The Group II antagonist, MCCG (200 μm), attenuated the frequency-dependent synaptic depression

The Group III antagonist, MSOP (3 mm), also attenuated the frequency-dependent synaptic depression. Figure 11A shows an example of the frequency-dependent depression before and in the presence of MSOP. As shown in the group data (Fig. 11B), MSOP, at the concentration that blocked the l-AP4-induced synaptic depression and spared the l-CCG-I-induced synaptic depression, also significantly attenuated the frequency-dependent depression (two-way ANOVA: P = 0.003, MSOP; P < 0.001, frequency; P = 0.145, interaction).

Figure 11
The Group III antagonist, MSOP (3 mm), attenuated the frequency-dependent synaptic depression

DISCUSSION

The major findings of the present study were that: (1) Group II and III but not Group I mGluR agonists reduced the amplitude of synaptically (ts) evoked EPSCs in NTS second-order neurones while having no significant effect on the amplitude of postsynaptically (AMPA)-evoked currents in the same neurones; (2) the genes for all eight mGluR subtypes were expressed in the cell bodies of the primary afferent autonomic fibres, a prerequisite for the receptors to be present at the central terminals; and (3) the frequency-dependent depression of synaptic transmission between primary autonomic afferent fibres and second-order NTS neurones, which has been documented as having a presynaptic mechanism (Felder & Heesch, 1987; Mifflin & Felder, 1988; Schild et al. 1995; Chen et al. 1999) was attenuated by the Group II and III antagonists. Together, these data are consistent with the hypothesis that activation of Group II and III mGluRs located on presynaptic terminals depresses autonomic signal transmission at NTS synapses.

In the present study, under voltage-clamp conditions to minimize the possible confounding influences of changes in the postsynaptic membrane potential, activation of Group II and III mGluRs depressed synaptic transmission between sensory afferent fibres and NTS second-order neurones in a concentration-dependent manner (Fig. 1 and Fig. 2). The findings suggest that as previously demonstrated in other neural networks (Scanziani et al. 1997; Cartmell & Schoepp, 2000), activation of mGluRs at NTS synapses regulates autonomic signal transmission by inhibiting glutamate release. Moreover, the apparent lack of effect of the Group I mGluR agonist also bears out the findings in other neural networks, suggesting that the Group I receptors may not regulate synaptic transmission (Pin & Duvoisin, 1995; Conn & Pin, 1997).

Two lines of evidence support the hypothesis that the synaptic depression was mediated by activation of mGluRs located on the presynaptic terminals of the primary afferent fibres. First, the synthetic machinery for the mGluRs was demonstrated in the cell bodies of the primary afferent fibres. Using real-time RT-PCR, which allows precise and sensitive quantification of PCR product (Pan et al. 2000), we detected gene expression for all eight mGluR subtypes in the cell bodies in the nodose and jugular ganglia (Fig. 5 and Fig. 6). While gene expression in the cell bodies does not guarantee insertion of the receptors into the presynaptic terminal, it is the first obligatory step; if there were no gene expression in the cell bodies, there could be no receptor at the central terminal. Second, agonist activation of Group II or III presynaptic mGluRs depressed the amplitude of synaptically evoked EPSCs, while having no consistent effect on the amplitude of postsynaptically (AMPA) evoked excitatory currents (Fig. 3 and Fig. 4); this also supports the hypothesis that the synaptic depression was caused by a decrease in synaptic glutamate release rather than by blockade of postsynaptic glutamate receptors or by some non-specific effect that decreased the excitability of the postsynaptic neurone.

Interestingly, the electrophysiology data showed that only the Group II and III receptors regulated synaptic transmission, despite the detection of message for all eight mGluR subtypes. The electrophysiological data also complement immunohistochemical data showing that mGluR 2 and 3 (Group II) and mGluR 7 (Group III) are predominantly expressed in fibres (putative axonal processes and terminals) in the NTS where they could readily modify synaptic transmission (Hay et al. 1999). There were, however, differences in the extent to which the Group II and III receptor agonists depressed synaptic transmission. The Group III agonist, l-AP4, reduced the amplitude of the synaptically evoked EPSCs by 100 % compared with 39 % by the Group II agonist, l-CCG-I. On the other hand, the concentration-response curve of l-AP4 spanned a three-log concentration and featured a high EC50 (1 mm), compared with an EC50 of 21 μm for l-CCG-I, a finding that could be explained by the 100-times lower affinity of l-AP4 for the Group II subtype, mGluR 7, compared with the affinity for the other Group II subtypes (mGluR 4, 6 and 8) (Okamoto et al. 1994).

Despite the large difference in the EC50 of the agonists, the results from the antagonist studies indicate that endogenously released glutamate activates both Group II and III receptors to reduce synaptic transmission (Fig. 10 and Fig. 11), suggesting that both Groups may modulate autonomic signal transmission in the behaving animal. It is tempting to speculate that lower frequencies of afferent input release sufficient glutamate to activate Group II (mGluR 2 and 3) and some Group III receptors (mGluR 4, 6 and 8), while at higher input frequencies, the greater glutamate spillover activates the Group III (mGluR 7) receptors to inhibit synaptic transmission to a greater extent. Alternatively, Cartmell and Schoepp, modelling data from a wide range of studies, suggested that each glutamate receptor subtype may have a distinctive geometric placement with respect to release sites on the presynaptic terminal, which would also influence the accessibility to endogenous glutamate release (Cartmell & Schoepp, 2000).

The antagonist studies further showed that the inhibitory effect of the Group II and III mGluRs on synaptic transmission was increasingly prominent with increasing frequencies of afferent input (up to 48 Hz) and was negligible at low input frequencies (< 9 Hz). One likely explanation is that with the higher frequencies of afferent input, the increased release of glutamate allows for further diffusion of the neurotransmitter in the synaptic cleft to access the presynaptic mGluRs. This concept is supported by electrophysiological evidence obtained in the NTS for a prolonged presence of glutamate in the synaptic cleft (Titz & Keller, 1997), favouring more extensive diffusion. This has also been shown to occur at mossy fibre synapses that also feature frequency-dependent activation of presynaptic mGluRs (Scanziani et al. 1997).

The NTS findings correspond to those in other CNS sites regarding the contribution of Group II and III presynaptic mGluRs to frequency-dependent synaptic depression (Dubé & Marshall, 2000). In the NTS, as the stimulation frequency was increased from 9 to 48 Hz, activation of Group II receptors contributed 13–34 % to the synaptic depression and activation of Group III receptors contributed 13–19 %. In the locus coeruleus, frequency-dependent depression was attenuated by 23 % by blocking Group III but not Group II receptors (Dubé & Marshall, 2000). In hippocampal mossy fibre-pyramidal cell synapses, the Group II/III antagonist, 4-carboxyphenylglycine (MCPG), actually increased synaptic transmission by 39 % at higher stimulation rates (1 Hz) while having no effect at low stimulation rates (0.05 Hz) (Scanziani et al. 1997). In the calyx of Held, Group II/III presynaptic mGluRs play a smaller role in frequency-dependent synaptic depression: the Group II/III antagonist, (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG) attenuated the synaptic depression by 10 % (von Gersdorff et al. 1997).

The question arises as to the physiological relevance of the modest contribution of mGluRs to frequency-dependent synaptic depression. In a previous study using curve-fitting analysis, we showed that frequency-dependent depression of baroreceptor afferent input to second-order NTS neurones in the intact animal limited the extent of the reflex output, that is the decrease in sympathetic nerve activity. The data predicted that prevention of a synaptic depression of 10 % in the NTS would allow for a 19 % greater reflex output, that is a 56 % increase in the reflexly evoked decrease in sympathetic nerve activity (Liu et al. 2000). Thus, activation of presynaptic mGluRs during high-frequency afferent activity may serve to limit excessive and perhaps unnecessary autonomic reflex outputs by limiting high-frequency signal transmission in the NTS.

Since mGluR activation only accounts for a portion of frequency-dependent synaptic depression in the NTS, other presynaptic mechanisms implicated elsewhere in the CNS may also contribute to the synaptic depression in the NTS. These mechanisms include the depletion of vesicles (Liu & Tsien, 1995) or the releasable pool of vesicles (Taschenberger & von Gersdorff, 2000), inactivation of presynaptic voltage-dependent Ca2+ channels (Wu & Saggau, 1997), and slow recovery of release probability following release of each quantum of transmitter (Silver et al. 1998). Even though in the present study, frequency-dependent depression in the NTS occurred in the absence of inhibitory GABAergic mechanisms, other studies have documented a frequency-dependent increase in GABA inhibition that may also contribute to synaptic depression in the intact animal (Brooks & Glaum, 1995; Glaum & Brooks, 1996; Grabauskas & Bradley, 1998). Such redundant mechanisms would confer both flexibility and dependability for synaptic modulation of afferent traffic at the very first synapse in the NTS under a variety of conditions. Finally, since recordings within the NTS were made in the caudomedial region where cardiovascular, gastrointestinal and respiratory neurones are located (Loewy, 1990), it seems likely that the mGluRs provide a general mechanism for regulating signal transmission in a variety of autonomic reflex pathways.

In conclusion, at early synapses in autonomic reflex pathways, Group II and III presynaptic mGluRs may provide a mechanism to dampen excessive, unnecessary amounts of glutamate released during high-frequency afferent input, thereby optimizing autonomic signal transmission at proximal synapses in the afferent pathway which may ultimately fine-tune autonomic reflex function.

Acknowledgments

The authors gratefully acknowledge the technical assistance of Judy Stewart. The authors thank Dr Shigetada Nakanishi for graciously providing the mGluR 6 clone. This work was supported by National Heart, Lung, and Blood Institute Grant HL-60560 and by the American Heart Association, Western States Affiliate Postdoctoral Fellowship 0020004Y.

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