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Group I Metabotropic Glutamate Receptors on Second-Order Baroreceptor Neurons Are Tonically Activated and Induce a Na⁺–Ca²⁺ Exchange Current

Department of Pharmacology, School of Medicine, University of California, Davis, California

Submitted 21 July 2005; accepted in final form 26 September 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The nucleus tractus solitarius (NTS) is essential for coordinating baroreflex control of blood pressure. The baroreceptor sensory fibers make glutamatergic synapses onto second-order NTS neurons. Glutamate spillover activates Group II and III presynaptic metabotropic glutamate receptors (mGluRs) on the baroreceptor central terminals to inhibit synaptic transmission, but the role of postsynaptic mGluRs is less understood. We used whole cell patch-clamping in anatomically identified second-order baroreceptor neurons in a brain stem slice to test whether Group I, II, and III mGluRs had postsynaptic effects at this first central synapse in the baroreceptor afferent pathway. The Group I agonist DHPG induced a depolarization and spiking that was mimicked by endogenous glutamate. Group I mGluR blockade prevented the depolarization and slightly hyperpolarized the neurons, suggesting a small tonic Group I mGluR activation. The DHPG-induced inward current consisted of voltage-dependent and -independent components; the former was blocked by TEA and the latter was blocked by replacing extracellular NaCl with LiCl or Tris-HCl. The DHPG current was potentiated in a Ca²⁺-free external solution and was diminished by intracellular dialysis with BAPTA and by perfusion with Na⁺–Ca²⁺ exchanger blockers, KB-R7943 or 3',4'-dichlorobenzamil. Intracellular dialysis with GDPS or heparin and perfusion with the PLC inhibitor U-73122 or the Ca²⁺-calmodulin inhibitor W-7 significantly decreased the DHPG current. The data suggest that Group I mGluRs on baroreceptor neurons are functional; are activated by endogenous glutamate; and activate a Na⁺–Ca²⁺ exchanger through G-protein, PLC, IP₃, and Ca²⁺-calmodulin mechanisms to excite the cell, thus providing postsynaptic mechanisms to enhance or prolong baroreceptor signal transmission.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The nucleus tractus solitarius (NTS) is the principal brain stem site for coordinating CNS control of blood pressure. Second-order NTS neurons are the first site of synaptic contact of the primary baroreceptor afferent fibers, where the blood pressure–related sensory information is first subject to neuromodulation. When a change in blood pressure occurs, the information is rapidly and efficiently transmitted to the central baroreflex network by glutamate binding to ionotropic glutamate receptors on the second-order baroreceptor neurons in NTS (Aylwin et al. 1997; Ohta and Talman 1994; Talman 1989). At these synapses, the information is fine-tuned by presynaptic and postsynaptic influences originating from local networks and other brain regions so that the baroreceptor signal output to sympathetic and parasympathetic premotoneuronal pools is optimized to regulate blood pressure. Dysregulation of baroreceptor signal transmission in the NTS disrupts cardiovascular homeostasis and contributes to the consequences of hypertension and heart failure (Timmers et al. 2004).

The G-protein–linked metabotropic glutamate receptors (mGluRs) provide acute and long-term modulation of glutamatergic transmission in various neural networks (Anwyl 1999). Eight subtypes of mGluRs divide into three groups based on signal transduction pathways, pharmacology, and genetic sequence. Group I mGluRs consist of mGluR1 and mGluR5 and are located largely postsynaptically where they couple to G_q/11-proteins to activate phospholipase C (PLC), mobilize intracellular Ca²⁺ and subsequently modulate various ion channels (Pin and Duvoisin 1995), although there is evidence that Group I mGluRs also act through G-protein–independent pathways (Heuss et al. 1999). Group II and III mGluRs are generally located presynaptically where they inhibit adenylyl cyclase, to inhibit synaptic transmission.

Hay et al. (1999) described the expression of four mGluR subtypes (mGluR1a, mGluR2/3, mGluR5, and mGluR7) within specific NTS subnuclei. However, the functional characterization of mGluRs in baroreceptor signal processing in the NTS has focused largely on Group II and III mGluRs, which suppress baroreceptor signal transmission by limiting glutamate release from primary baroreceptor afferent fibers (Chen et al. 2002; Liu et al. 1998) and Group II mGluRs to decrease -aminobutyric acid (GABA) release (Chen and Bonham 2004). The role of the postsynaptic mGluRs in regulating baroreceptor signaling in the NTS is poorly understood. Matsumura et al. (1999) showed that NTS microinjection of the Group I mGluR antagonist, 1-aminoindan-1,5-dicarboxylic acid (AIDA) in vivo resulted in an 8 mmHg increase in blood pressure and a 28% increase in sympathetic nerve activity; these findings suggest that Group I mGluRs may provide a low level tonic activation of the NTS baroreceptor pathway to augment baroreflex control of blood pressure. Still the cellular effects of postsynaptic mGluR activation, whether postsynaptic mGluRs are activated by endogenous glutamate, or the underlying ionic mechanisms have not been determined on NTS second-order baroreceptor neurons.

Thus we asked the following questions: 1) are Group I mGluRs functional on second-order baroreceptor neurons, 2) are they stimulated by endogenous glutamate, and 3) what are the ionic mechanisms and signaling pathways? Because neurons in various autonomic pathways are intermingled in the NTS, we used whole cell patch-clamping on second-order NTS baroreceptor neurons anatomically identified by the presence of attached boutons of the primary baroreceptor fibers to specifically determine the contribution of mGluRs to baroreceptor signal transmission.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
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 the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Labeling second-order baroreceptor NTS neurons

Male Sprague–Dawley rats (11 wk old, 322 ± 7 g) were anesthetized by intramuscular injection of ketamine (40 mg kg^–1) and xylazine (8 mg kg^–1). As previously described (Chen et al. 2002, 2004), 4- to 5-mm segments of both aortic depressor nerves (ADNs) were carefully isolated and placed on a section of parafilm for application of the crystal form of the fluorescent tracer, 1,1'-dilinoleyl-3,3,3',3' tetra-methylindocarbo-cyanine, 4-chlorobenzenesulfonate [FAST DiI solid; DiI ^9,12-C₁₈(3)] on these nerves and coated with polyvinylsiloxane gel. DiI is transported anterogradely to label the terminal boutons, without being transported transynaptically. To allow for transport of the dye to the terminal boutons, the rats were allowed to recover for 2 wk before the experimental protocols were performed (Chen et al. 2002, 2004).

Brain stem slice preparation and electrophysiology

Rats were anesthetized with a combination of ketamine (20 mg kg^–1) and xylazine (2 mg kg^–1) and decapitated. As in previous studies (Chen et al. 2002, 2004) the brain was rapidly exposed and submerged in ice-cold (<4°C) high-sucrose artificial cerebrospinal fluid (aCSF) that contained (in mM): 3 KCl, 2 MgCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 10 glucose, 220 sucrose, and 2 CaCl₂ (300 mosmol kg^–1); the pH was 7.4 when continuously bubbled with 95% O₂-5% CO₂. Brain stem coronal slices (250 µm thick) containing the intermediate to caudal NTS and the tractus solitarius (TS) were cut with a Vibratome 1000 (Technical Products International, St. Louis, MO). After incubation for 45 min at 37°C in high-sucrose aCSF, the slices were placed in normal aCSF that contained (in mM): 125 NaCl, 2.5 KCl, 1 MgCl₂, 1.25 NaH₂PO₄, 25 NaHCO₃, 25 glucose, and 2 CaCl₂ (300 mosmol kg^–1); the pH was 7.4 when continuously bubbled with 95% O₂-5% CO₂. Four or five slices were obtained from each animal. During the experiments, a single slice was transferred to the recording chamber, held in place with a silk mesh, and continuously perfused with oxygenated aCSF at a rate of approximately 4 ml min^–1. All experiments were performed at 33–34°C.

All recordings were taken from second-order NTS baroreceptor neurons with attached ADN boutons. The neurons were visualized with Nomarski infrared differential interference contrast (IR-DIC) and the fluorescent boutons were visualized with an optical filter set for DiI (XF108, Omega Optical, Brattleboro, VT) and an image-integrating system (InstaGater, Dage-MTI, Michigan City, IN). All images were captured with a charge-coupled device (CCD) camera (CCD-100, Dage-MTI) displayed on a TV monitor and stored in a PC computer using Computer Eyes software (Winnov, Sunnyvale, CA). Recordings were made with the Axopatch 1D patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 2 kHz, digitized at 10 kHz with the DigiData1322A interface (Axon Instruments), and stored in an IBM-compatible computer. Data were analyzed off-line using the pCLAMP9 software (Axon Instruments). After establishing the cell-attached configuration with a seal resistance of >1 G, whole cell currents were recorded using borosilicate glass pipettes (2.2–5 M; 3.5 ± 0.8 M, mean ± SD) filled with a potassium gluconate (K-gluconate)–based solution containing (in mM): 130 KC₆H₁₁O₇, 1 NaCl, 1 MgCl₂, 2 K-ATP, 0.3 Na–guanosine-5'-triphosphate (GTP), 10 EGTA, and 10 Hepes (300 mosmol kg^–1); pH was adjusted to 7.4 with KOH. In some experiments, to examine intracellular signaling pathways, Na-GTP was replaced with 1 mM of the nonhydrolyzable guanosine-5'-O-(2-thio-diphosphate) (GDP) analog, GDPS, to determine G-protein participation; EGTA was replaced with the Ca²⁺ chelator BAPTA (20 mM) to determine the involvement of intracellular Ca²⁺; and the inositol-1,4,5-triphosphate (IP₃) receptor antagonist heparin (300 units ml^–1) was added to the normal pipette solution to test for Ca²⁺ release by IP₃ receptors. The series resistance was <25 M (12.4 ± 5.3 M, mean ± SD). After establishing the whole cell configuration, neurons were tested for TS input with the membrane voltage clamped at –60 mV. All experiments were performed with 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(t)quinoxaline-7-sulfonamide disodium salt (NBQX, 10 µM), D-2-amino-5-phosphonopentanoic acid (AP-5, 50 µM), and the GABA_A receptor antagonist (–)-bicuculline methiodide (bicuculline, 10 µM) to exclude ionotropic glutamate and GABA_A receptor inputs under current-clamp conditions. Tetrodotoxin (TTX, 1 µM) was added to block synaptic transmission under voltage-clamp conditions.

Agonist studies

Selective mGluR agonists (S)-3,5-dihydroxyphenylglycine (3,5-DHPG, Group I mGluR, 30 or 100 µM) and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an agonist with selectivity for mGluR5 subtype of Group I; (2S,3S,4S)-CCG/(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I, Group II mGluR, 20 or 100 µM) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP4, Group III mGluR, 300 or 1,000 µM) were tested under current-clamp and voltage-clamp conditions. Drugs were added to perfusion for 1 min in a random order. For Group I mGluRs, concentration-dependent responses were examined with the DHPG (1, 3, 10, 30, 100 µM) or CHPG (30, 100, 300, 1,000 M) applied in a random order.

Endogenous glutamate release

To increase glutamate in the cleft to test for endogenous glutamate activation of mGluRs, a glutamate transporter inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, 250 µM), was added to the perfusion during current-clamp recordings. In pilot studies, PDC efficacy in increasing glutamate in the cleft was verified on second-order baroreceptor NTS neurons by the following: 1) PDC in the perfusate increased the decay time constant for TS-evoked glutamatergic excitatory postsynaptic currents by 34% (2.7 ± 0.3 to 3.7 ± 0.6 ms, n = 3); and 2) increased the peak amplitude of the inward current evoked by application of exogenous glutamate (300 µM) with NBQX, AP5, bicuculline, and TTX in the perfusate by 146% (146.3 ± 39.8 pA, n = 4 vs. 59.5 ± 7.2 pA, n = 5; one-tail t-test, P = 0.034), consistent with previous findings (Carter and Regehr 2000). The neurons were initially held at their own resting membrane potentials (–50.5 ± 1.3 mV) and then recorded in current-clamp mode. After 5-min perfusion of PDC, the Group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) was perfused for 1 min to observe any voltage changes.

Data analysis

Data are expressed as means ± SE unless otherwise stated. Peak voltage or current changes were obtained from the greatest changes in either direction from a stable control level (30 s before any drug application) for current- or voltage-clamp recordings, respectively. Differences among the mGluR agonists were compared with a one-way ANOVA followed by the Fisher's least-significant difference (LSD) post hoc test when appropriate. Differences before, during, and after Group I mGluR antagonists were compared with a one-way repeated-measures ANOVA followed by the Fisher's LSD post hoc test. DHPG and CHPG concentration–response curves were fitted to a logistic function to obtain the EC₅₀ and EC₉₀. DHPG-induced inward currents were detected and comparisons before and during changes in external or internal solution were made with paired t-test. Values of P < 0.05 were considered significant.

Drugs

Stock solutions of U-73122, U-73343, KB-R7943, 3',4'-dichlorobenzamil (DCB), thapsigargin, KN-62, staurosporine, and PMA (4,9,12,13,20-pentahydroxytiglia-1,6-dien-3-one-12-myristate-13-acetate) were made with DMSO. All other drugs were dissolved in aCSF just before application. Ketamine and xylazine were obtained from Vedco (St. Joseph, MO). DiI was obtained from Molecular Probes (Eugene, OR). Polyvinylsiloxane gel was obtained from Charlisle Laboratories (Rockville Center, NY). 3,5-DHPG, L-CCG-I, L-AP4, CHPG, AIDA, KB-R7943, U-73122, U-73343, KN-62, W-7, staurosporine, and PMA were obtained from Tocris (Ballwin, MO). K-Gluconate, bicuculline, Mg-ATP, Na-ATP, EGTA, HEPES, CaCl₂, TEA, DCB, thapsigargin, NBQX, AP-5, TTX, and GDPS were obtained from Sigma (St. Louis, MO). All other chemicals were obtained from Fisher (Fair Lawn, NJ).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Whole cell patch recordings were performed on a total of 157 second-order baroreceptor NTS neurons, which were identified by the presence of fluorescent boutons and were located at 0 to 1,000 µm caudal to obex in the NTS. Figure 1A shows one such neuron viewed under infrared differential interference contrast (IR-DIC), then under fluorescence to visualize presynaptic baroreceptor afferent fiber terminal boutons (Fig. 1B), as an overlay of fluorescence and IR-DIC images (Fig. 1C) and with the patch electrode attached to cell for whole cell recording. All results were obtained in the presence of the ionotropic glutamate receptor antagonists NBQX (10 µM) and AP-5 (50 µM) and the GABA_A receptor antagonist bicuculline methiodide (10 µM), to exclude ionotropic glutamate and GABA_A receptor inputs. In voltage-clamp experiments, TTX (1 µM) was additionally perfused.

View larger version (119K): [in this window] [in a new window]   FIG. 1. Second-order baroreceptor nucleus tractus solitarius (NTS) neuron. A: NTS neuron viewed under infrared differential interference contrast (IR-DIC). Calibration bar = 20 µm. B: same neuron viewed under fluorescence to visualize presynaptic baroreceptor afferent fiber terminal boutons. C: overlay of fluorescence and IR-DIC. D: patch electrode attached to cell for whole cell recording.

We determined the effect of the Group I agonist DHPG, the Group II agonist L-CCG-I, and the Group III agonist L-AP4 on changes in whole cell membrane potential and current in anatomically identified second-order baroreceptor neurons. As shown in the voltage trace (Fig. 2A), DHPG (30 µM) depolarized the neuron that then fired a burst of action potentials. In the same neuron L-CCG-I (20 µM) had a very small depolarizing effect, whereas L-AP4 (300 µM) did not significantly change the membrane potential.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Group I, II, or III metabotropic glutamate receptor (mGluR) agonist effects on baroreceptor neurons under current- and voltage-clamp conditions. A: current-clamp recording. Baroreceptor neuron was initially held at –60 mV. (S)-3,5-Dihydroxyphenylglycine (DHPG, 30 µM) significantly depolarized the neuron, which fired a burst action potentials. (2S,3S,4S)-CCG/(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I, 20 µM) slightly depolarized it and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4, 300 µM) had no effect. B: group data confirmed that DHPG significantly depolarized the neurons, whereas in the same neurons L-CCG-I elicited a small depolarization and L-AP4 had no consistent effect (*P < 0.05). C: voltage-clamp recording. Reflecting the current-clamp recordings, DHPG (30 µM) evoked an inward current, whereas L-CCG-I (20 µM) and L-AP4 (300 µM) had no effect in the same cell as A. D: group data showing that DHPG evoked an inward current, whereas L-CCG-I and L-AP4 did not (*P < 0.05).

The DHPG-induced inward current was concentration dependent with an EC₅₀ of 9.1 µM and an EC₉₀ of 32.6 µM (Fig. 3A). The more selective mGluR5 agonist CHPG also evoked an inward current in a concentration-dependent manner with an EC₅₀ of 337.1 µM and an EC₉₀ of 545.2 µM. The Group I mGluR antagonist AIDA, tested at three concentrations (0.3, 1.0, and 3.0 mM), attenuated the DHPG (30 µM)–induced inward current in a concentration-dependent manner (Fig. 3, B and C).

View larger version (13K): [in this window] [in a new window]   FIG. 3. DHPG- and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG)–evoked inward currents and dose relationships and effects of Group I mGluR antagonist on the current. A: both the DHPG- and CHPG-induced peak currents increased in a dose-dependent manner. Number in parentheses indicates number of neurons. B: example traces of DHPG application before and during 1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) perfusion. AIDA pretreatment significantly attenuated the DHPG-induced inward current. C: group data of AIDA effects on the DHPG-induced inward current confirmed that AIDA antagonized the Group I mGluR excitatory effects in a concentration-dependent manner.

The physiological relevance of the excitatory effect of the Group I mGluRs in modulating baroreceptor signal transmission depends on whether they are activated by endogenous glutamate. As shown in Fig. 4A when endogenous glutamate was increased in the synaptic cleft with PDC (250 µM), the neuron depolarized and then fired a burst of action potentials. Perfusion with the Group I mGluR antagonist AIDA (1 mM) not only blocked the depolarization but also resulted in a hyperpolarization. PDC depolarized all 11 neurons; in three of the 11 neurons, the depolarization led to abundant action potential firings; in all three neurons the action potential firings were in bursting patterns (Fig. 4A, bottom), consistent with action of Group I mGluRs in other networks (Prisco et al. 2002; Zheng and Johnson 2002). After antagonist washout, the neuron again depolarized and spiked. The findings were consistent in all neurons tested (n = 11). As shown in the group data (Fig. 4B), PDC depolarized the neurons (3.7 ± 1.1 mV) and blockade of Group I mGluRs with AIDA not only abolished the depolarization but also resulted in a 6.6 ± 1.9 mV hyperpolarization, which reversed with washout of the antagonist. The findings suggest that the Group I mGluRs can be tonically activated if there are sufficient levels of endogenous glutamate in the perisynaptic cleft (n = 11; one-way repeated-measures ANOVA, P < 0.001; Fisher's LSD, P < 0.05).

View larger version (30K): [in this window] [in a new window]   FIG. 4. Endogenous glutamate activates postsynaptic Group I mGluRs on NTS baroreceptor neurons. A: neurons were initially held at their own resting membrane potential (Vm), and then current-clamp recordings were started (in this case resting Vm = –49 mV). Ionotropic GluR and -aminobutyric acid type A (GABAA)–Rs were blocked (top bar). Addition of L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, 250 µM, middle bar) initially slightly hyperpolarized and then gradually depolarized the neuron, which then fired action potentials (suggesting depolarizing action of increased Glu in the cleft). Group I mGluR antagonist, AIDA (1 mM, bottom bar) not only abolished the depolarization but also slightly hyperpolarized the neuron. After washout of AIDA, the neuron again showed depolarization and spiking. Note that the spiking pattern during PDC perfusion shows an oscillatory pattern (bottom left). B: group data showing depolarization during endogenous Glu release with PDC, blockade of depolarization and hyperpolarization during AIDA, and after washout. Baseline Vm was before PDC perfusion. Data indicate that endogenous Glu can stimulate NTS baroreceptor neurons through Group I mGluRs (*P < 0.05).

The relationship of the DHPG-induced current to membrane voltage was obtained using a slow-voltage ramp-down protocol from +40 to –150 mV, 2-s duration from the holding potential of –60 mV before and at the peak of the DHPG-induced current (Fig. 5A). The DHPG-induced current was obtained by subtracting the control current from the current obtained during DHPG taken at voltage steps of 0 to –120 mV. The averaged slopes of the I–V plots at resting membrane potentials were parallel (7.8 ± 1.2 and 8.1 ± 1.2 nS, respectively; n = 24, paired t-test, P = 0.379), indicating that the slope conductance was not changed by DHPG (Fig. 5B). The averaged subtraction currents indicate that the DHPG-induced inward current was relatively independent of membrane voltage at potentials negative to –60 mV, but strongly voltage dependent at potentials positive to –60 mV (Fig. 5C). Because Group I mGluR activation has been reported to inhibit the voltage-sensitive K⁺ current (I_K) (Charpak et al. 1990; Hay and Lindsley 1995; Lüthi et al. 1996; Schrader and Tasker 1997), we compared the DHPG-induced I–V relationship in the absence and presence of I_K blocker TEA. A 10 mM concentration of TEA, shown to attenuate I_K by 85% in NTS neurons (Moak and Kunze 1993), attenuated the negative slope region of the DHPG-induced I–V relationship (Fig. 6B), suggesting that K⁺ currents are involved in mediating the Group I mGluR response at voltages positive to –60 mV; however, DHPG could still induce an inward current at –60 mV (Fig. 6A), which showed a relative voltage independence over almost the entire voltage range (Fig. 6B). This I–V relationship was also observed when DHPG was applied at different holding potentials (–90, –60, –30, or 0 mV) at which voltage-sensitive currents were mostly inactivated (Fig. 6C). The following experiments focused on the voltage-independent component of the inward current.

View larger version (19K): [in this window] [in a new window]   FIG. 5. Effect of voltage on Group I mGluR agonist DHPG-induced inward current. A, top: ramp protocol. Bottom: example trace of the whole cell recording at a –60 mV holding potential; the ramp protocol was performed just before DHPG was perfused (gray arrow) and just after (black arrow). Bar above the current trace indicates DHPG (30 µM) application, which induced an inward current that reached a maximum plateau level that was sustained during the second ramp protocol performed at the point of black arrow. B: averaged I–V relationships before (gray dots) and during (black dots) DHPG (n = 24). Error bars are omitted for clearer view. Outward currents were activated at depolarizing potentials to –60 mV to a greater extent before DHPG. Averaged slopes (dI/dV) of the I–V plots near the holding potential were not different. C: subtraction current from traces in B. Error bars are also omitted. Inward currents were independent of membrane voltage at potentials negative to –60 mV, but strongly voltage dependent at potentials positive to –60 mV.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Effect of tetraethylammonium (TEA) on DHPG-induced I–V relationships. A: example traces of the DHPG-induced current in the absence and presence of TEA (10 mM). Ramp protocols were performed at arrows. Note that TEA did not affect the DHPG-induced current amplitude (68.2 pA before TEA vs. 73.1 pA during TEA in the example; 76.5 ± 22.6 and 61.3 ± 11.8 pA in the absence and presence of TEA, respectively, n = 3, paired t-test, P = 0.431; data not shown). B: averaged subtraction currents (n = 3) in the absence (gray) and presence (black) of TEA. Error bars on subtraction currents are omitted for clearer view. Voltage dependency of the inward currents at potentials positive to –60 mV was diminished by TEA, suggesting that the voltage-dependent component is most likely a result of inhibitory action of DHPG on K+ currents. Voltage-independent component was unchanged by TEA. C: DHPG-induced inward current at different holding potentials. Data were well fitted by a standard straight line, which indicated a reversal potential of 55 mV (fitted line was not shown).

To determine the ionic mechanism(s), we evaluated the DHPG-induced current with ion substitutions. As shown in the time control, repeated applications of DHPG evoked inward currents of the same magnitude (Fig. 7A). The first DHPG response was 66.3 ± 12.7 pA and the second DHPG response was 64.5 ± 12.9 pA (n = 15, paired t-test, P = 0.584). To test the involvement of Na⁺, we replaced external NaCl with LiCl (125 mM) or Tris-HCl (Fig. 7B). Replacing Na⁺ with Li⁺ nearly abolished the DHPG-induced inward current (23.5 ± 5.2% of control, n = 6, paired t-test, P = 0.023), as did replacing Na⁺ with Tris-HCl (Fig. 7C) (17.4 ± 2.5% of control, n = 5, paired t-test, P = 0.024). Because Tris or Li⁺ cannot substitute for Na⁺ in the Na⁺–Ca²⁺ exchanger, but can substitute for Na⁺ for the nonselective cationic channel (Fraser and MacVicar 1996; Kakehata et al. 1993; Zhainazarov and Ache 1995), these findings suggested a role for the Na⁺–Ca²⁺ exchange current. We then tested the involvement of extracellular Ca²⁺. The DHPG-induced current was enhanced when Ca²⁺ was removed from the extracellular solution (nominally Ca²⁺-free solution) in the presence of normal Mg²⁺ (Fig. 7D) (253.8 ± 41.7% of control; n = 6, paired t-test, P = 0.024), suggesting that the DHPG-induced current was sensitive to extracellular Ca²⁺. The inhibition of the inward current by extracellular Ca²⁺ is similar to findings by others (Ishibashi et al. 2003; Okada et al. 1999).

View larger version (22K): [in this window] [in a new window]   FIG. 7. DHPG-induced current in different extracellular solutions. Bars above traces indicate 1-min application of DHPG (30 µM). All extracellular solutions contained ionotropic and GABAA receptor antagonists and tetrodotoxin (TTX). All example traces (left trace in each pair of traces) were control DHPG responses in normal artificial cerebrospinal fluid (aCSF). Bar between traces represents 50 pA. A: time control experiments. DHPG (30 µM) was applied twice with intervals of more than 5 min. DHPG-induced current was the reproducible. B and C: effects of extracellular Na+-free solution on the DHPG-induced current. When NaCl in aCSF was replaced by either LiCl or Tris-HCl, the DHPG-induced current was almost abolished [*P = 0.023 (Li: n = 6) and P = 0.024 (Tris: n = 5) with paired t-test]. D: effects of extracellular Ca2+-free solution on the DHPG-induced current. DHPG-induced current became greater in Ca2+-free solution (*P = 0.024), indicating Ca2+ inward currents may not contribute directly to the DHPG-induced inward current.

View larger version (58K): [in this window] [in a new window]   FIG. 8. Intracellular Ca2+ dependency of the DHPG-induced current. A: cell attached configuration with the first (1) and second (2) pipette viewed under IR-DIC. Calibration bar = 20 µm. Presynaptic baroreceptor afferent fiber terminal boutons were visualized under fluorescence and confirmed from overlay image of fluorescence and IR-DIC (3). B: time control experiments with different pipettes. Bars above traces indicate 1-min application of DHPG (30 µM). All extracellular solutions contained ionotropic and GABAA receptor antagonists and TTX. Between 2 recordings of the DHPG-induced current, the first patch pipette was pulled out of the cell and then the whole cell configuration was established with the second pipette, which was almost identical to the first one. Changing the pipette took at least 4 to 5 min and, consequently, the second DHPG application was performed about 10 min after the first application. Note that the current amplitude was not affected by changing the pipette (n = 6, paired t-test, P = 0.299). B: effects of fast-acting Ca2+ buffer, BAPTA (20 mM). As in A, the recording pipette was changed to another one but containing BAPTA. DHPG-induced current was inhibited with the BAPTA-containing pipette (*P = 0.021). Combined with the data in Fig. 7D, the results suggest that DHPG-induced current might be dependent on the Ca2+ concentration difference across the cell membrane.

View larger version (24K): [in this window] [in a new window]   FIG. 9. Effects of Na+–Ca2+ exchanger inhibitors on the DHPG-induced current. Figure presentation is the same as in Fig. 7. Bars above traces indicate 1-min application of DHPG (30 µM). All extracellular solutions contained ionotropic and GABAA receptor antagonists and TTX. Bar between traces represents 50 pA. A: effects of amiloride analog, 3',4'-dichlorobenzamil (DCB, 100 µM). DHPG-induced current was diminished in the presence of DCB (*P = 0.008). B: effects of the Na+–Ca2+ exchanger inhibitor, KB-R7943 (100 µM). DHPG-induced current was nearly abolished in the presence of KB-R7943 (*P = 0.027).

Because Group I mGluRs have been shown to act through G-protein–dependent and –independent pathways, we next determined whether the DHPG-induced inward current depended on activation of G-proteins. Blockade of G-protein function by incorporating the nonhydrolyzable GDP analog GDPS (1 mM) into the second pipette solution in which Na-GTP was omitted, significantly inhibited the DHPG-induced inward current by 86.4 ± 5.4% (Fig. 10A, n = 6, paired t-test, P = 0.018), suggesting that activation of Group I mGluRs on baroreceptor neurons is G-protein mediated. Because the Group I mGluR-associated G-protein–dependent pathway couples to PLC, we tested the effect of the PLC inhibitor, U73112 [GenBank] (10 µM) and its inactive analog U73343 [GenBank] (10 µM). Pretreatment with U73112 [GenBank] significantly attenuated but did not abolish the DHPG-induced current (52.2 ± 11.8% of control, n = 7, paired t-test, P = 0.009) (Fig. 10B), whereas U73343 [GenBank] had no effect (99.3 ± 6.3% of control, n = 4, paired t-test, P = 0.776) (Fig. 10C).

View larger version (26K): [in this window] [in a new window]   FIG. 10. Involvements of G-protein and phospholipase C on the DHPG-induced inward current. Figure presentation is the same as in Fig. 7. Bars above traces indicate 1-min application of DHPG (30 µM). All extracellular solutions contained ionotropic and GABAA receptor antagonists and TTX. Bar between traces represents 50 pA. A: inhibition of DHPG-induced current by a nonhydrolyzable guanosine-5'-O-(2-thio-diphosphate) (GDP) analog, GDPS. As in Fig. 8, the recording pipette was changed between DHPG applications; in this case the second pipette contained GDPS (1 mM). DHPG-induced current was clearly inhibited with intracellular GDPS, suggesting that the current is G-protein dependent (*P = 0.018). B and C: effects of PLC inhibitor. Although extracellular application of the membrane permeable PLC inhibitor U73122 [GenBank] (10 µM) reduced the amplitude of the DHPG-induced current (*P = 0.009), pretreatment with the same concentration of U73343 [GenBank] , the inactive analog of U73122 [GenBank] , did not change the DHPG-induced current.

View larger version (25K): [in this window] [in a new window]   FIG. 11. Effects of antagonism of inositol-1,4,5-triphosphate (IP3) receptors, Ca2+-ATPase, calmodulin (CaM), and CaM-dependent protein kinase II on the DHPG-induced inward current. Figure presentation is the same as in Fig. 7. Bars above traces indicate 1-min application of DHPG (30 µM). All extracellular solutions contained ionotropic and GABAA receptor antagonists and TTX. Bar between traces represents 50 pA. A: effects of IP3 receptor antagonist heparin (300 units ml–1). As in Fig. 8, the recording pipette was changed before the second DHPG application; in this case the second pipette contained heparin. DHPG-induced current was clearly inhibited by intracellular application of heparin (*P = 0.048), suggesting the involvement of IP3 receptors. B: effects of the Ca2+-ATPase inhibitor, also known as the IP3-independent Ca2+ release inhibitor thapsigargin (3 µM). DHPG-induced current was not affected by pretreatment of thapsigargin. C: contribution of Ca2+/CaM on the DHPG-induced current. W-7 (100 µM) significantly attenuated the DHPG-induced inward current observed under the control condition (*P = 0.002). D: effects of Ca2+/CaM-dependent protein kinase II inhibitor KN-62 (3 µM) on the DHPG-induced current. KN-62 had no effect on the DHPG-induced inward current.

View larger version (26K): [in this window] [in a new window]   FIG. 12. No effect of the protein kinase C (PKC) modulators on DHPG-induced currents. Figure presentation is the same as in Fig. 7. Bars above traces indicate 1-min application of DHPG (30 µM). All extracellular solutions contained ionotropic and GABAA receptor antagonists and TTX. Bar between traces represents 50 pA. A: effects of the PKC inhibitor staurosporine (1 µM). DHPG-induced current was still clearly observed after staurosporine treatment. B: effects of the PKC enhancer (4,9,12,13,20-pentahydroxytiglia-1,6-dien-3-one-12-myristate-13-acetate) (PMA, 3 µM). Pretreatment of PMA did not change the DHPG-induced current amplitude. Combined with data in A, the results suggest that PKC did not contribute to the DHPG-induced current.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

DHPG excites second-order baroreceptor neurons by a direct postsynaptic mechanism

The Group I agonist DHPG induced a depolarization and spiking in the presence of ionotropic GluR and GABA_A-R antagonists. In the presence of TTX both DHPG, which binds to Group I mGluR1 and mGluR5 and CHPG, the more selective subgroup 5 agonist evoked inward currents in a concentration-dependent manner. The single study localizing mGluRs in the NTS by immunohistochemistry reported the presence of both mGluR5 and mGluR1a subtypes in the intermediate and caudal NTS, where second-order baroreceptor neurons are located; however, no mGluR1a immunoreactivity was observed in the medial aspects of these NTS regions, which may account for the dominant contribution of the mGluR5 agonist to the response (Hay et al. 1999).

The DHPG-induced influx of positive charges consisted of at least two components: a voltage-independent component, likely resulting from activation of the Na⁺–Ca²⁺ exchanger; and a voltage-dependent component at more positive voltages, which was TEA sensitive and possibly resulting from inhibition of K⁺ current. Although the voltage-dependent, TEA-sensitive component was not the focus of this study, it is worth noting that both components may contribute to baroreceptor neuronal excitability. For example, at near resting conditions, postsynaptic mGluR activation would likely depolarize the baroreceptor neuron by the exchanger, whereas at more depolarized states the voltage-dependent, TEA-sensitive mechanism might be more prominent in contributing to the excitability.

Physiological relevance

Baroreceptor afferent input to the central baroreflex network regarding changes in blood pressure is rapidly and efficiently transmitted by glutamate binding to ionotropic glutamate receptors on the second-order baroreceptor neurons. The slower depolarization of the neurons by glutamate binding to the Group I mGluRs may either enhance or prolong the excitability of the second-order baroreceptor neurons by interactions with other postsynaptic processes, for example, by amplifying the impact of other excitatory inputs that might otherwise be without effect or by suppressing inhibitory inputs. In the present study, when endogenous glutamate was increased in the synaptic cleft in the presence of blockade of the ionotropic glutamate receptors and of GABA receptors, the neurons spontaneously depolarized and in some cases fired a burst of action potentials, mimicking the effects of the Group I mGluR agonist. Moreover, blockade of Group I mGluRs prevented the depolarization and resulted in hyperpolarization, suggesting that with sufficient amounts of glutamate in the cleft, Group I mGluRs can provide a small tonic increase in background excitability of the second-order neurons. This result builds on findings that microinjection of the Group I antagonist AIDA in the NTS of rats in vivo resulted in an increase in blood pressure and an increase in sympathetic nerve activity (Matsumura et al. 1999). Thus sufficient levels of glutamate in the NTS may tonically activate Group I mGluRs to provide a low-level background increase in excitability of NTS neurons in the baroreceptor network, which in turn tonically suppresses the sympathetic nervous system to decrease blood pressure. The source of glutamate, aside from the primary baroreceptor afferent fibers, may also originate from other brain regions involved in neural regulation of cardiovascular function, including the area postrema (Chen and Bonham 1998), hypothalamic defense area (Silva-Carvalho et al. 1995), and periaqueductal gray (Boscan and Paton 2005).

Contribution of the Na⁺–Ca²⁺ exchange current

The DHPG current consisted of a voltage-independent component at voltages negative to –60 mV and a voltage-dependent component at positive voltages. The voltage-dependent component of the DHPG-induced was attenuated by TEA, but the DHPG-induced inward current remained and was the focus of this study.

The ion-substitution studies and ion channel blocker results support the involvement of a Na⁺–Ca²⁺ exchanger in the DHPG current: First, the DHPG-induced current was reduced when Na⁺ was replaced by Li⁺ or Tris. Neither can substitute for Na⁺ in the Na⁺–Ca²⁺ exchanger, in contrast to the nonselective cationic channels that are permeable to both Li⁺ and Tris (Crepel et al. 1994; Fraser and MacVicar 1996; Kakehata et al. 1993; Partridge and Swandulla 1988; Yellen 1982; Zhainazarov and Ache 1995). Second, the current was enhanced in nominally free Ca²⁺, characteristic of Na⁺–Ca²⁺ exchange currents (Ishibashi et al. 2003; Okada et al. 1999; Umezu et al. 2004; Wu et al. 2004). Third, the DHPG-induced current was attenuated when intracellular Ca²⁺ was buffered with BAPTA, suggesting a dependency on intracellular Ca²⁺. Although the Ca²⁺-activated nonselective (CAN) cationic current is also sensitive to internal BAPTA, its permeability to Li⁺ or Tris distinguishes its contribution to the DHPG-induced current in these neurons. Finally, the current was attenuated by the Na⁺–Ca²⁺ exchanger blocker, DCB or KB-R7943, at a concentration similar to that used in other studies showing inhibition of the exchanger (Burdakov et al. 2003; Eriksson et al. 2001; Iwamoto and Shigekawa 1998).

The contribution of the Na⁺–Ca²⁺ exchanger to Group I mGluR-activated inward currents has not been broadly reported, being demonstrated with the broad spectrum agonist 1S,3R-ACPD in only two types of neurons: rat basolateral amygdale neurons by Keele and colleagues (1997) and ventromedial hypothalamic neurons by Lee and Boden (1997) and suggested by Linden et al. (1994) in studies of mouse and cerebellar Purkinje neurons (Staub et al. 1992). Other mechanisms mediating mGluR-induced cationic currents include Ca²⁺-dependent or -independent selective cation currents (Congar et al. 1997; Crepel et al. 1994; Guerineau et al. 1995; Zheng et al. 1995), voltage-dependent Ca²⁺ channels, and Ca²⁺-sensitive transient receptor potential (TRP)–like currents (Gee et al. 2003). Other studies have linked Group I mGluR activation to inhibition of K⁺ currents, including the delayed outward rectifier (I_K) (Charpak et al. 1990; Hay and Lindsley 1995; Lüthi et al. 1996; Schrader and Tasker 1997), resting K⁺ current (Guerineau et al.1994; Schrader and Tasker 1997), and slow Ca²⁺-dependent K⁺ current (I_AHP) (Abdul-Ghani et al. 1996a,b; Gerber et al. 1992; Schrader and Tasker 1997). There are no previous data on postsynaptic Group I mGluR effects on NTS baroreceptor neurons, although a study in dissociated heterogeneous NTS neurons reported that Group I mGluRs may also depolarize NTS neurons by facilitating the L-type channel or inhibiting the N/P/Q-type Ca²⁺ channels (Endoh 2004). Whether these mechanisms operate in baroreceptor neurons is not clear, but our finding that removal of extracellular Ca²⁺ augmented the DHPG-induced inward current suggests that voltage-dependent Ca²⁺ mechanisms may not contribute to the depolarization of second-order baroreceptor neurons, at least in the slice. One other study that focused on presynaptic effects of mGluRs in heterogeneous NTS neurons, either dissociated or in a slice, reported no postsynaptic effects of Group I mGluR activation based on no change in the holding current after application of a single dose (10 µM) of DHPG (Jin et al. 2004). This dose was close to the ED₅₀ (9.1 µM) in our study, which evoked currents of about 40 pA. Although it is difficult to compare responses to a single dose, there were other differences between the two studies that could have affected the mGluR responses: 1) the present recordings were specifically taken from baroreceptor second-order neurons, whereas the previous studies were conducted on heterogeneous second-order NTS neurons; 2) recordings in the present study were undertaken in neurons held at body temperature, whereas neurons in the previous study were undertaken at room temperature; and 3) the present data were obtained from adult rats (13 wk), whereas data from the previous study were obtained from rats aged between 2 and 8 wk. In any event, the diversity of ion channels and signal transduction pathways activated by the Group I mGluRs in various neural networks may help to explain the diversity of responses mediated by these receptors, depending on the cell type and function.

G-protein–signaling pathways

Although Group I mGluRs have been shown to involve G-protein–independent pathways in some neurons, they are largely known to release Ca²⁺ from intracellular stores in a Gq/G11–PLC–IP₃ pathway (Heuss et al. 1999). In the present study, the GTP analog GDPS abolished the DHPG current; the PLC inhibitor significantly attenuated it, whereas the inactive enantiomer had no effect; and blockade of an increase in intracellular Ca²⁺ with IP₃ receptor blockade with heparin significantly attenuated the inward current. The finding that the PLC inhibitor U-73122, although significantly attenuating, did not abolish the DHPG current could be interpreted to suggest that another pathway was involved. However, a comparison of U-73122 effects in slice preparations (Lee and Boden 1997) and dissociated neurons (Ishibashi et al. 2003) shows that the inhibitory effect is smaller in slice preparations even with higher concentrations. Furthermore, our unpublished data using membrane permeable BAPTA (BAPTA-AM, 100 µM) also resulted in an unremarkable blockade (32%) of the DHPG current. Together, these findings raise the possibility that drug permeability might be diminished in slice preparations. Importantly, however, U73343 [GenBank] , the inactive analog of U73122 [GenBank] , had no effect on the DHPG-induced current, indicating that the U73122 [GenBank] attenuation of the inward current was unlikely mediated through some nonspecific mechanism. Evidence in support of Ca²⁺ release through the IP₃ receptors was the finding that preventing an intracellular rise Ca²⁺ by blocking the Ca²⁺-ATPase with thapsigargin had no effect. However, we cannot rule out the possibility that thapsigargin may not have depleted Ca²⁺ stores because Ca²⁺ was not actively released (e.g., by brief depolarization of the neurons with high K⁺ in advance). Nevertheless these findings suggest that the intracellular pathway between the mGluRs and the Na⁺–Ca²⁺ exchanger is the classic G-protein–PLC–IP₃ receptor pathway. The attenuation of the DHPG current by the CaM antagonist W-7 indicates that the mGluR-induced Ca²⁺ mobilization stimulates the Ca²⁺/CaM signaling pathway, although not through a Ca²⁺/CaM-dependent protein kinase II because KN-62, the potent Ca²⁺/CaM-dependent protein kinase II, had no effect on the DHPG current.

The findings are consistent with the following events: activation of the Group I mGluRs triggers the G-protein–PLC–IP₃ receptor pathway. The resultant increase in intracellular Ca²⁺ activates a Ca²⁺/CaM pathway followed by activation of a Na⁺–Ca²⁺ exchanger or alternatively by direct activation of the exchanger, resulting in an influx of 3 Na⁺ with an efflux of 1 Ca²⁺. The latter possibility is supported by the finding that the exchanger possesses CaM binding sites in an intracellular loop (Li et al. 1991).

A characteristic of the mGluRs is the modest changes in neuronal or synaptic excitability; however, even modest changes in excitability in the NTS are important in shaping baroreceptor signal transmission. In a previous study, in which we simultaneously recorded second-order NTS baroreceptor neuronal and sympathetic nerve activity in vivo, the data predicted that a 10% decrease in the NTS output (spike frequency) of the baroreceptor signal could lead to a 20% reduction in sympathoinhibition (Liu et al. 2000).

In summary, the findings provide new evidence for functional excitatory postsynaptic Group I mGluRs on anatomically identified second-order baroreceptor neurons that activate Na⁺–Ca²⁺ exchange current through a G-protein–PLC–IP₃ pathway.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-60560.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
The authors gratefully acknowledge Drs. Chao-Yin Chen and Hiroaki Misonou for helpful criticisms in preparing the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. C. Bonham, School of Medicine, 4150 V Street, 1104 PSSB, University of California, Davis, Medical Center, Sacramento, CA 95817 (E-mail: ann.bonham{at}ucdmc.ucdavis.edu)

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