Role of glutamate in a visceral sympathoexcitatory reflex in rostral ventrolateral medulla of cats
Abstract
The rostral ventrolateral medulla (rVLM) is involved in processing visceral sympathetic reflexes. However, there is little information on specific neurotransmitters in this brain stem region involved in this reflex. The present study investigated the importance of glutamate and glutamatergic receptors in the rVLM during gallbladder stimulation with bradykinin (BK), because glutamate is thought to function as an excitatory neurotransmitter in this region. Stimulation of visceral afferents activated glutamatergic neurons in the rVLM, as noted by double-labeling with c-Fos and the cellular vesicular glutamate transporter 3 (VGLUT3). Visceral reflex activation significantly increased arterial blood pressure as well as extracellular glutamate concentrations in the rVLM as determined by microdialysis. Barodenervation did not alter the release of glutamate in the rVLM evoked by visceral reflex stimulation. Iontophoresis of glutamate into the rVLM enhanced the activity of sympathetic premotor cardiovascular rVLM neurons. Also, the responses of these neurons to visceral afferent stimulation with BK were attenuated significantly (70%) by blockade of glutamatergic receptors with kynurenic acid. Microinjection of either an N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonopentanate (25 mM, 30 nl) or an dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (2 mM, 30 nl) into the rVLM significantly attenuated the visceral sympathoexcitatory reflex responses. These results suggest that glutamate in the rVLM serves as an excitatory neurotransmitter through a baroreflex-independent mechanism and that both NMDA and AMPA receptors mediate the visceral sympathoexcitatory reflex responses.
activation of sympathetic visceral chemo- and mechanosensitive afferents evokes excitatory cardiovascular reflex responses, including increases in blood pressure and heart rate consequent to sympathoadrenal activation (12, 35, 39). These autonomic responses to visceral sympathetic afferent stimulation collectively are referred to as the visceral sympathoexcitatory reflex (38). The rostral ventral lateral medulla (rVLM), a component of many cardiovascular reflex pathways associated with sympathetic excitation, is considered to be a major center involved in processing this reflex (15, 25, 38). For example, anatomical evidence has demonstrated that stimulation of cardiac sympathetic afferents activates neurons within the rVLM (22). Also, direct electrical or chemical stimulation of neurons in the rVLM increases sympathetic nerve activity and arterial blood pressure (15). Furthermore, stimulation of splanchnic nerves increases the discharge of cardiovascular premotor neurons in the rVLM and evokes excitatory cardiovascular responses (58, 59, 61). Conversely, lesion of neurons in the rVLM abolishes somatic pressor reflexes (52).
Although several neurotransmitters and/or modulators, including glutamate, catecholamines, GABA, acetylcholine, angiotensin, serotonin, and opioids, may be involved in regulating activity of neurons in the rVLM (13, 28, 29, 47, 49, 51) and ultimately sympathetic activity and cardiovascular function (15, 26, 53, 54), glutamate likely is the principal excitatory neurotransmitter in the rVLM (15, 17). In this regard, some sympathetic premotor neurons within the rVLM contain glutamate (44). Microinjection of glutamate into the rVLM directly excites neurons in the rVLM leading to increases in sympathetic nerve activity and arterial blood pressure (16). In contrast, microinjection of kynurenic acid, a nonspecific glutamate ionotropic receptor antagonist (13), into the rVLM diminishes the blood pressure response to static muscle contraction (7). Additionally, Ishide and his colleagues (28) have observed that extracellular glutamate concentrations in the rVLM are increased during the exercise pressor reflex in rats. However, there is no information regarding the role of endogenous glutamate in regulating sympathetic premotor neurons within the rVLM during visceral sympathoexcitatory reflexes.
The physiological effects of glutamate in tissues are mediated, in part, by two glutamate ionotropic receptors, N-methyl-d-aspartate (NMDA) and non-NMDA dl-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). Previous studies have demonstrated that pulsatile compression of the rVLM increases sympathetic outflow and arterial blood pressure by activating rVLM neurons through stimulation of both NMDA and AMPA receptors (43). Other studies have documented that blockade of both NMDA (4, 48) and AMPA receptors (33, 37) in the rVLM attenuates muscle contraction-evoked excitatory cardiovascular reflex responses. However, the relative importance of two ionotropic glutamate receptors in the rVLM during visceral sympathoexcitatory reflex remains poorly defined.
The purpose of the present study was to evaluate the importance of endogenous glutamate and glutamatergic receptors within the rVLM during a well-characterized visceral sympathoexcitatory reflex involving stimulation of chemosensitive receptors in the gallbladder with bradykinin (BK). We hypothesized that glutamate serves as an excitatory neurotransmitter during visceral sympathoexcitatory reflexes. As such, stimulation of visceral sympathetic afferents induces an increase in glutamate release, which activates sympathetic premotor neurons in the rVLM through stimulation of glutamate ionotropic receptors, ultimately leading to an excitatory cardiovascular reflex response. A preliminary report of this study has been published (19).
MATERIALS AND METHODS
General Surgical Preparation
The experimental preparations and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of the University of California, Irvine. Studies were performed on cats of either sex (2.5–4.5 kg). Animals were anesthetized initially with ketamine (40–50 mg/kg im) and α-chloralose (50 mg/kg iv) and were instrumented for recording blood pressure and heart rate as described previously (12, 35). Systemic blood pressure was measured with a cannula inserted into the femoral artery, which was connected to a pressure transducer (Statham P23 ID). Body temperature was monitored continuously with a rectal probe and was maintained between 37.0° and 38.5°C with a water-perfused heating pad and an external heat lamp.
Barodenervation.
The ventral surface of the neck was exposed by a midline incision. The carotid sinus and cervical vagii were separated from the internal carotid and common carotid arteries, respectively, and sectioned distally to denervate baroreceptor afferents. Barodenervation was verified by the absence of a decrease in heart rate in response to an increase in arterial blood pressure induced by an administration of phenylephrine (10 μg/kg iv, GensiaSicor Pharmaceuticals, Irvine, CA).
Double-Fluorescent Immunohistochemical Labeling with c-Fos and VGLUT3 in rVLM
We examined for evidence of colocalization of c-Fos and glutamate in rVLM neurons after BK stimulation of the gallbladder. Vesicular glutamate transporters (VGLUTs) specifically transport glutamate into vesicles of neurons and, therefore, offer a marker to distinctively identify neurons that use glutamate as a neurotransmitter (56).
As described in our previous studies (22, 23), animals were perfused transcardially with 0.9% normal saline and ice-cold 4% paraformaldehyde in 0.1 M PBS, 90 min after completion of experimental procedures described below. The medulla oblongata was harvested and stored in 4% paraformaldehyde for 2 h and in 30% sucrose for 48 h. Coronal sections of the brain (30 μm) were made with a cryostat microtome (Leica CM 185 Heidelberger Strasse, Nussloch, Germany) and collected serially in cold cryoprotectant solution (11).
After being rinsed in 0.1 M PBS (pH = 7.4), brain sections were placed in 1% normal donkey serum (Jackson, West Grove, PA) for 1 h. The tissues were incubated in the PBS solution containing two primary antibodies, i.e., mouse monoclonal anti-c-Fos (1:2,000 dilution, Santa Cruz Biotechnology) and guinea-pig polyclonal anti-VGLUT3 (1:5,000, Chemicon International, Temecula, CA) at 4°C for 48 h. Sections then were treated with two secondary antibodies raised in the donkey, including rhodamine-conjugated anti-mouse and fluorescein-conjugated anti-guinea pig, at 4°C for 24 h. The slides were coverslipped using mounting medium (Vector). Immunohistochemical control studies were performed by the omission of the primary and secondary antibodies.
Brain sections were scanned and examined with a standard fluorescent microscope (Nikon, E400, Melville, NY). We selected four sections that most closely matched two standard stereotaxic planes (P10.0 and P10.8) of Berman's atlas for the cat, two sections for each plane, which displayed the rVLM. Selected sections containing fluorescent activity were evaluated with a laser scanning confocal microscope (Zeiss LSM 510, Meta System, Thornwood, NY). The number of single- and double-labeled cells was counted on both the right and left sides of brain sections in each animal. The average number of labeled cells in the rVLM was calculated by dividing the total number of cells by four, representing the number of sections used for cell counting.
Microdialysis and HPLC Measurement of Glutamate
The cat's head was fixed in a stereotaxic apparatus (Kopf Instruments), and a craniotomy was performed to expose the ventral surface of the medulla for microdialysis. For placement of probes into the rVLM, the basioccipital bone was removed from the atlanto-occipital membrane with rongeurs and the craniotomy extended for ∼4 mm on each side of the midline over the medullary surface, as described in our previous study (12). The dura was cut and the cerebrospinal fluid removed to expose the medullary surface. Gel foam was used to minimize bleeding during this procedure.
After surgery, two microdialysis probes (CMA/12, 1 mm length, 0.5 mm diameter, CMA, Acton, MA) were placed bilaterally in the rVLM with the use of a stereotaxic carrier (Kopf Instruments). Each probe was positioned at the surface of the medulla at a point overlying the paragigantocellularis lateralis, as indicated by axis coordinates: 2.5–3.5 mm lateral to the midline, 0.5–1 mm below the ventral surface of the medulla, and 0.5–1.5 mm caudal to the trapezoid border (12, 20). Extracellular fluid collected for 10 min (1.5 μl/min, total 15 μl) was sampled continuously with a refrigerated fraction collector in each protocol and stored at −80°C until the assay was performed with artificial cerebral spinal fluid (CSF; pH 7.4). The CSF contained 0.2% bovine serum albumin, 0.1% bacitracin, and the following ions (in mM): 6.2 K+, 134 Cl−, 2.4 Ca2+, 150 Na+, 1.3 P−, 13 HCO3−, and 1.3 Mg2+. Confirmation of the accurate placement of the microdialysis probes into a pressor region of the brain stem was verified by noting an immediate increase in blood pressure after injection of 30 nl of 1 mM l-glutamate (RBI, Natick, MA) into the area.
Glutamate in the microdialysate was measured as described previously (41). Thus 15 μl of microdialysate were mixed with 1.5 μl of o-phihalic dicarboxaldehyde (OPA) solution (20 mg of OPA, 0.5 ml of 1 M sodium sulfite, and 10 ml of 4 M sodium borate solution) for precolumn derivation for 25 min at 25°C (30). The mixed sample was analyzed immediately thereafter for glutamate. The mobile phase for isocratic elution of glutamate consisted of 100 mM NaH2PO4 (pH 5.8), 10% methanol, and 0.5 mM Na2EDTA, which was run through a Spherisorb PS Phase Separation ODS2, 5 μm HPLC analytical column. The mobile phase was pumped (510 Pump, Waters, Milford, MA) at a flow rate of 1.0 ml/min. Glutamate was detected coulometrically by a Coulochem II detector (ESA, Chelmsford, MA) with a 5020 guard cell and a 5011 analytic cell (ESA). The applied working potentials were 910 mV for the guard cell and 310 mV (E1) and 860 mV (E2) for the analytic cell. The detection limit for this assay was 50 pg for glutamate. Confirmation of peak identity was accomplished by concurrently running samples with known standards.
Extracellular Neuronal Recording With Iontophoresis
To confirm the neuronal responses of premotor neurons that receive inputs from visceral afferents in the rVLM to exogenous glutamate, we conducted extracellular neuronal recordings during iontophoresis of glutamate into the rVLM. Extracellular neuronal recording in the rVLM and iontophoresis techniques have been described previously (59).
Briefly, the splanchnic nerve was stimulated with 0.4–0.6-mA, 0.5-ms pulses at 2 Hz. The intermediolateral (IML) column was located by electrical stimulation (0.1–0.4 mA, 2 Hz, 0.5 ms duration) with concentric bipolar electrodes with 200 μm OD (Frederick Haer) to evoke a small reproducible increase in blood pressure of 5–10 mmHg to confirm proper location of the stimulating electrode for collision testing. Microiontophoretic experiments utilized multibarrel glass microelectrodes containing monofilament glass fibers to record the extracellular potentials from single neurons and to apply drugs at the recording site. One barrel contained 0.5 M sodium acetate and 2% Chicago sky blue (Sigma, St. Louis, MO), whereas three other barrels were filled with glutamate (20 mM), kynurenic acid (50 mM, pH 7.5), and NaCl solution (4 M). A platinum electrode was used for recording. A positive current of 120 and 230 nA was used for 4-min iontophoresis of glutamate and kynurenic acid, respectively; a negative current of 5–10 nA was used to prevent leakage of drugs. Current balancing was accomplished by filling one of the barrels with 4 M NaCl. Action potentials were amplified with a preamplifier (Grass P511) attached to a high-impedance probe (Grass H1 P5) and were filtered (0.3–10 kHz) and monitored with an oscilloscope (Tectronix 2201).
The relationship between neuronal activity and blood pressure was assessed by time domain analysis using arterial pulse-triggered averaging. The relationship between these two events also was assessed by frequency domain analysis using coherence analysis (6, 59).
Brain Histology
The location of each extracellular neuronal recording, microdialysis probe, and microinjection site in the rVLM was determined at the end of each experiment by injecting 50 nl of 0.5% pontamine blue dye (Chicago Sky Blue) into the brain. The brain stem was removed and fixed in 4% paraformaldehyde and 20% sucrose for at least 24 h. Frozen coronal 60-μm sections were cut with a microtome cryostat. The sites of injection, identified by the atlas of Berman (8), were reconstructed from the dye spots.
Experimental Protocols
Expression of c-Fos and VGLUT3 in rVLM.
After barodenervation, vagotomy and laparotomy to provide exposure of the gallbladder, animals were allowed to stabilize for 4 h. A flank incision provided access to the gallbladder as described previously (12, 35). The gallbladder was rinsed with saline after the peak pressor response, ∼1 min after application of BK (5–10 μg). Animals were divided randomly into two groups: BK (1–10 μg in 50 μl; Sigma, n = 6) was applied to the gallbladder six times over a 100-min period with 20-min intervals between each application (1st though 6th stimulation). c-Fos gene expression is normally induced within 30 min after the activation of a neuron. The protein product of the Fos gene reaches maximal expression 60–90 min after stimulation and remains elevated for 2–5 h (34). The smallest dose of BK that induced at least a 30-mmHg increase in mean arterial pressure was chosen. In a sham-operated control group (n = 4), a similar procedure was performed using the vehicle for BK, 0.9% saline. Blood pressure and heart rate were monitored throughout the experiment and were recorded continuously during the period of application of BK or 0.9% saline. Tissue from the rVLM was harvested for c-Fos and VGLUT3 immunohistochemical labeling.
Glutamate release in rVLM during visceral reflex stimulation with and without barodenervation.
After insertion of the microdialysis probes, the animal was allowed to stabilize for 1 h. Preliminary study demonstrated that 1 h was sufficient to stabilize the extracellular glutamate in the rVLM (see results). During this period, CSF was continuously dialyzed at 1.5 μl/min, while alternating 10-min collections of dialysates from each probe (volume, 15 μl) for each collecting period were performed to establish a stable baseline (control) concentration of extracellular glutamate. BK (10 μl/ml) then was applied to the serosal surface of the gallbladder with a 1-cm2 pledget of filter paper soaked with BK. The filter paper was removed after the cardiovascular responses (∼2–3 min), and the gallbladder was washed at least three times with saline to remove excess BK. The order of procedure was two successive control collection periods to evaluate stability of basal glutamate, followed by an application of BK to the gallbladder at the beginning of a 10-min collection period. Seven animals were used in this protocol.
In another six barodenervated cats, animals were stabilized for a period of 1 h after denervation. Extracellular glutamate in the rVLM was collected after the same procedures as mentioned above. Furthermore, in five other cats, the glutamate was collected before and during intravenous infusion of phenylephrine hydrochloride (0.5 mg/ml in saline, Sigma) at a rate of 3–5 ml/h to increase blood pressure by >30 mmHg for ∼2–3 min to test the influence of the arterial baroreflex on rVLM glutamate release.
Response of rVLM premotor sympathetic neurons to glutamate.
We used the following procedures to examine responses of rVLM premotor neurons to glutamate and glutamatergic receptor blockade. First, the IML was stimulated continuously at 2 Hz. Neurons in the rVLM that responded to stimulation of the IML were tested for criteria that indicated antidromic activation, as described previously (58, 59). Second, cardiovascular characteristics of these rVLM neurons were examined after an administration of nitroglycerin (5 mg/ml). Third, glutamate was iontophoresed onto each neuron for 4 min, followed by kynurenic acid for 4 min followed by repeat glutamate iontophoresis in five cats. Neuronal activity in the rVLM at baseline and during reflex excitation consequent to a stimulation of the splanchnic nerve was recorded after glutamate, kynurenic acid, and repeat glutamate administration.
Blockade of NMDA and AMPA receptors in rVLM.
Placement of glass micropipettes (30-μm tip diameter) for microinjection followed the same procedure as described in the microdialysis section above. Six cats received microinjection of normal saline into the rVLM as a vehicle control. Twelve cats were used to determine the role of NMDA and AMPA receptors in the visceral excitatory reflex responses with NMDA [2-amino-5-phosphonopentanate (AP-5), n = 6] or AMPA receptor blockade [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), n = 6]. After observing two repeatable blood pressure responses to BK stimulation, we unilaterally microinjected normal saline (0.9%, 30 nl), AP-5 (25 mM, 30 nl) (60), or CNQX (2 mM, 30 nl) (42) into the rVLM ∼2 min before the third BK stimulation. Responses in blood pressure and heart rate were recorded before, during, and after BK stimulation.
Statistical analysis.
Data are expressed as means ± SE. We used the Kolmogorov-Smirnoff test to determine whether the data were normally distributed. Baseline and peak values of neuronal activity, blood pressure, and glutamate concentrations were analyzed using a one-way repeated-measures ANOVA, followed by comparison for individual differences using the Tukey test. Comparison between control and BK stimulation for immunohistochemical study was made with the Student's t-test. Values were considered significantly different when P < 0.05. All statistical calculations were performed with a statistical software package (SigmaStat for Windows, version 3.0, Jandel Scientific Software, San Rafael, CA).
RESULTS
Expression of c-Fos and Glutamatergic Neurons in rVLM
We found that VGLUT3 and c-Fos positive neurons were distributed bilaterally in the rVLM in both control and BK-treated groups. It was noted that there were more VGLUT3-containing neurons in this area compared with c-Fos immunoreactive nuclei (Table 1). Most c-Fos nuclei were in cells containing VGLUT3. When compared with neurons of control animals (n = 4), neurons labeled with c-Fos and c-Fos + VGLUT3 were significantly increased (P < 0.05) in the rVLM of BK-treated cats (n = 6; Table 1). The majority of cells (80%) responding to BK stimulation were glutamatergic. Figure 1 \. shows confocal micrographs of neurons double labeled with c-Fos and VGLUT3 in the rVLM from a cat treated with BK.
Fig. 1.Confocal microscopic images of vesicular glutamate transporters 3 (VGLUT3) and c-Fos immunoreactivity in rostral ventrolateral medulla (rVLM; +2.7 mm rostral to obex). A and B: immunostaining for VGLUT3 (green) and c-Fos (red), respectively. C: merged images from A and B. Arrows in A, B, and C indicate neurons containing VGLUT3, c-Fos, and VGLUT3 + c-Fos, respectively.
Release of Glutamate in rVLM During Visceral Sympathoexcitatory Reflex With and Without Barodenervation
Chromatographic spectra of glutamate in standards and microdialysates collected from one animal detected by coulometric detector with HPLC are displayed in Fig. 2. We noted sharp peaks from glutamate in both microdialysates, which were verified with authentic standard. The glutamate peak in the microdialysate (Fig. 2C) during BK-evoked visceral sympathoexcitatory reflex responses is larger than the prestimulation peak (Fig. 2B).
Fig. 2.Glutamate levels within the rVLM before and during bradykinin (BK) stimulation on the gallbladder measured by microdialysis and HPLC/electrochemical detection. HPLC traces show glutamate peaks of standard (0.5 μM of glutamate in artificial cerebrolspinal fluid; A) and microdialysis samples at baseline (0.24 μM; B) and during BK stimulation (0.41 μM; C). Detection was accomplished coulometrically. Limit of detection of glutamate was 50 pg.
In a preliminary study we observed that the concentration of glutamate stabilized 60 min after insertion of microdialysis probes into the rVLM (0.39 ± 0.05 vs. 0.41 ± 0.06 μM, 60 vs. 70 min after probe insertion, n = 4). Therefore, we chose to assess the concentration of glutamate in microdialysates collected between 60 and 70 min after probe insertion as the prestimulation level. The microdialysate concentrations of glutamate were significantly increased during BK stimulation of visceral sympathetic afferents compared with those at the prestimulation level (i.e., control) in seven cats (Fig. 3C). We also observed that BK stimulation of visceral afferents significantly elevated mean arterial blood pressure compared with prestimulation (Fig. 3, A and B). In contrast, when the microdialysis probes were placed inadvertently in an area outside of the rVLM in five cats, despite the fact that BK stimulation significantly increased mean arterial pressure to 148 ± 5 mmHg from the prestimulation level of 110 ± 4 mmHg, microdialysate concentrations of glutamate during and after BK stimulation were not increased (0.22 ± 0.04 vs. 0.23 ± 0.03 μM, BK stimulation vs. prestimulation).
Fig. 3.Influence of visceral afferent stimulation on cardiovascular responses and rVLM glutamate with and without barodenervation. A: reflex increase in arterial blood pressure (BP) evoked by application of BK on gallbladder in intact (left) and barodenervated (right) cat. Arrow indicates application of BK. B: peak changes in mean arterial pressure (MAP) during application of BK in intact (n = 7) and denervated animals (n = 5). C: BK stimulation increased glutamate in rVLM. There was no significant difference in reflex increase in MAP and glutamate release between the intact and barodenervated animals. Values are means ± SE. *P < 0.05 compared with values before BK (control).
In five barodenervated cats, BK stimulation of the gallbladder also significantly increased the mean arterial blood pressure and glutamate concentration compared with cats at prestimulation levels (Fig. 3). The magnitude of increases in both glutamate concentrations and blood pressure induced by BK stimulation was not significantly different than these observed in the baroreceptor intact group (glutamate, 0.10 ± 0.03 vs. 0.11 ± 0.05 μM; and arterial pressure, 31 ± 7.8 vs. 41 ± 8.7 mmHg, intact vs. barodenervation, P > 0.05, Fig. 3). Additionally, we observed that intravenous injection of phenylephrine significantly increased mean blood pressure by 33 ± 6.5 mmHg but did not alter the glutamate concentration (0.41 ± 0.03 vs. 0.43 ± 0.03 μM, baseline vs. phenylephrine, n = 5) in five other denervated cats.
Glutamatergic Mechanisms of rVLM Premotor Sympathetic Neurons
We studied five rVLM premotor neurons with an average baseline activity of 0.9 ± 0.2 impulses/s (imp/s). Each neuron could be driven antidromically from the IML of the spinal cord. Figure 4A displays an example of the collision test used, in part, to define rVLM neurons that were stimulated antidromically and, hence, could be classified as premotor sympathetic. The antidromically induced spike collided with the action potential evoked by stimulation of the splanchnic nerve. In this example, the distance from the IML to the rVLM was 8.4 cm, the latency was 15.8 ms, and the calculated conduction velocity (CV) was 5.3 m/s. For the group of five neurons, the IML-rVLM distance was 8.9 ± 0.6 cm, the latency was 16.3 ± 1.2 ms, and the CV was 5.5 ± 0.9 m/s. These five neurons were also examined with respect to their response to baroreceptor input. Altered baroreceptor input to the premotor sympathetic neurons after administration of nitroglycerin, which lowered mean blood pressure from 121 ± 4.5 to 76 ± 3.5 mmHg, increased discharge activity from 1.1 ± 0.3 to 2.7 ± 0.6 imp/s. Each neuron responding to antidromic stimulation of IML was also examined by several methods with respect to its relationship to cardiovascular-related activity. Discharge frequencies of the neurons were subjected to 1) frequency domain analysis using coherence to evaluate their discharge relationship to the cardiovascular blood pressure activity and 2) time domain analysis with arterial pulse-triggered averaging. We found a strong correlation with average coherence of 0.88 at a frequency 2.5 ± 0.5 Hz (heart rate, 150 ± 4.1 beats/min) between neural activity and arterial blood pressure. Arterial pulse-triggered averaging likewise showed a strong relationship between the discharge rate of the five neurons and arterial blood pressure. An example of a neuron with coherence of 0.9 at a frequency of 2.6 Hz is shown in Fig. 4C. Figure 4, B and D, respectively, shows the arterial pulse-triggered analyses and the change of neuronal activity after administration of nitroglycerin.
Fig. 4.Frequency and time domain analyses of relationship between discharge of rVLM neuron and arterial BP and effect of glutamate on rVLM neuronal activity. A: antidromic collision of a premotor rVLM neuron (latency, 15.8 ms; and refractory period, 5.2 ms) during antidromic stimulation of intermediolateral column (IML) at T2 of spinal cord. In first sweep (a), neuron was activated by stimulation of splanchnic nerve and IML. Second sweep (b) shows collision as we reduced interval between orthodromic and antidromic electrical IML stimulus. In third sweep (c), antidromic spike reappeared as interval was increased. Critical interval (latency + refractory period) was 21 ms. Time of stimulation of the splanchnic nerve (↓) and IML (*) are indicated. B: BP and rVLM neuronal response to administration of nitroglycerin, demonstrating that this neuron is a cardiovascular excitatory neuron. imp/s, Impulses/s. C: arterial pulse-triggered analysis of rVLM activity (average based on 100 trials; bin widths of 12 ms). D: frequency domain analysis of autospectra (AS) of BP and rVLM neuronal activity and corresponding coherence function. Coherence of 0.9 occurred at a frequency of 2.6 Hz (heart rate, 156 beats/min).
We observed that iontophoresis of glutamate significantly increased the spontaneous neuronal activity of premotor sympathetic cardiovascular neurons in the rVLM from 0.8 ± 0.2 to 2.8 ± 0.2 imp/s (P < 0.05), while iontophoresis of kynurenic acid blocked the rVLM neuronal responses to exogenous glutamate (Fig. 5A). Kynurenic acid also significantly inhibited rVLM neuronal activity evoked by splanchnic nerve stimulation from 33 ± 6.6 to 10 ± 1.7 imp/30 stimulations (P < 0.05, Fig. 5B). However, kynurenic acid did not alter baseline activity of rVLM neurons, suggesting that glutamate does not contribute to tonic activity in this brain stem region.
Fig. 5.Neuronal responses of premotor sympathetic cardiovascular neurons in the rVLM to glutamate (Glu). Spontaneous neuronal responses of premotor neurons to repeated iontophoresis of glutamate and effect of ionotropic receptor blockade with kynurenic acid (Kyn) on glutamate response (A). Effect of kynurenic acid on splanchnic nerve evoked rVLM neuronal activity (B). *P < 0.05 compared with values before iontophoresis of glutamate. †P < 0.05 compared with values after iontophoresis of glutamate. ‡P < 0.05 compared with values before iontophoresis of kynurenic acid. pre and post, Before and after administration of kynurenic acid.
Blockade of NMDA and AMPA Receptors in rVLM
In the control group of six animals, the reflex increase in blood pressure evoked by repeated BK stimulation was consistent during two repeated stimulations, whereas microinjection of saline did not alter the reflex responses (Fig. 6A). Microinjection of the NMDA receptor antagonist AP-5 into the rVLM of six cats significantly attenuated the reflex responses to BK stimulation from 26 ± 5 to 6 ± 2 mmHg (P < 0.05, Fig. 6B). Microinjection of the AMPA receptor antagonist CNQX into the rVLM of six separate cats also significantly attenuated the blood pressure responses from 25 ± 4 to 4 ± 2 mmHg (P < 0.05, Fig. 6C).
Fig. 6.Effect of antagonism of ionotropic glutamate receptors on visceral reflex responses to repeated BK stimulation. A: reflex increase in arterial BP evoked by repeated BK stimulation, followed by microinjection of saline into the rVLM. Bar graphs show peak changes in MAP during BK stimulation before and after blockade of NMDA receptor [2-amino-5-phosphonopentanate (AP-5); B] and AMPA receptor [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); C]. 1st and 2nd, Repeated control stimulations of gallbladder with BK or without intervention. Values are means ± SE. *P < 0.05 compared with values before BK (baseline). †P < 0.05 compared with vehicle control. n, Number of animals.
Histology
Figure 7 illustrates the brain histological data. Among the 35 injections that went into the rVLM area, 5 injection sites were related to iontophoresis of glutamate and kynurenic acid, 12 injection sites to microdialysis in the rVLM, and 18 injection sites to microinjection of NMDA or AMPA blockade. Five microdialysis sites were found to be outside the rVLM.
Fig. 7.Composite map showing locations of the injection sites in and around rVLM. Injection sites for iontophresis (▴), microdialysis (*, within rVLM; and •, outside rVLM), and microinjection (+) are shown. Sections represent combinations of medullary regions rostral to the obex. Inferior olivary nucleus (ION), alaminar spinal trigeminal nucleus (5SP), and retrofacial nucleus (RFN) are shown for reference.
DISCUSSION
This is the first study to demonstrate that stimulation of visceral spinal afferents activates sympathetic premotor neurons by releasing glutamate in the rVLM, which acts through both NMDA and AMPA glutamatergic ionotropic receptors to induce excitatory cardiovascular responses. Stimulation of visceral afferents activated glutamatergic neurons in the rVLM, assessed by double-labeling with c-Fos and VGLUT3. Visceral reflex activation significantly increased the extracellular concentration of glutamate in the rVLM. Blockade of either NMDA or AMPA receptors of neurons in the rVLM significantly attenuated the visceral pressor reflex responses evoked by BK-gallbladder stimulation. We also found that iontophoresis of glutamate into the rVLM increased activity of sympathetic premotor cardiovascular neurons in the rVLM. Furthermore, the response of these neurons to visceral afferent stimulation was attenuated by blockade of glutamatergic receptors with kynurenic acid. Taken together, these data suggest that glutamate acting through NMDA and AMPA receptors in the rVLM serves as an important excitatory neurotransmitter in processing the visceral excitatory cardiovascular reflex responses.
Previous work from this laboratory has shown that the rVLM is involved in processing visceral sympathoexcitatory reflexes during BK stimulation of thoracic and abdominal visceral spinal afferents (12, 22, 58, 59). We have demonstrated that both opioid and nonopioid neurotransmitters participate in prolonged inhibition of these visceral reflexes by somatic afferent stimulation (12, 14, 36). However, the neurotransmitters in this region that are involved in visceral excitatory reflexes have not been established. In this respect, the immediate response of rVLM neurons receiving convergent input from visceral and somatic afferents is excitation rather than inhibition, the latter typically requiring 10–15 min of prolonged somatic stimulation to observe (59).
In the present study, we observed that neurons responding to stimulation of visceral sympathetic afferents express the VGLUT3. We selected VGLUT3 as the marker for glutamatergic neurons because identification of VGLUT3 represents a valuable method to visualize the perikarya of glutamatergic neurons in the brain (18, 50) compared with other VGLUT isoforms (VGLUT1 and VGLUT2), which are located in neuronal process other than cell bodies (24). These data suggest that this group of neurons activated by input from visceral afferents is glutamatergic.
Increases in extracellular glutamate in the rVLM evoked by muscle contraction are associated with reflex pressor responses, whereas decreases in glutamate release in the rVLM are associated with reflex depressor responses (27, 28, 31, 37). We observed an increase in rVLM extracellular glutamate associated with pressor responses during BK-gallbladder stimulation, suggesting that glutamate may serve as an excitatory neurotransmitter in visceral sympathoexcitatory reflexes. In contrast to studies of somatic afferent stimulation (2), we found that the glutamate release evoked by visceral afferent stimulation was independent of baroreceptor activation.
There has been substantial discussion about the role of extracellular glutamate concentrations with respect to its function in synaptic (neural) transmission. Some have argued that extracellular glutamate sampled by microdialysis originates not from extracytotic (vesicular) release from the synaptic cleft but rather from sources that are tetrodotoxin and calcium independent, i.e., from glial cells, and is therefore not related to neurotransmission (57). However, this concept has been successfully challenged recently with the in vivo demonstrations that vesicular glutamate is released from glial cells and, through a calcium-dependent mechanism, contributes to synaptic activity via cystine-glutamate antiporter exchange (5, 9, 45, 46). It is clear that there is cross talk between neuronal and nonneuronal (glial) elements through release and reuptake and that both contribute to extracellular amino acid concentrations. Surrounding neurons and glia contribute to the basal levels of glutamate sampled by microdialysis. Increases above basal levels occurring during evoked activity as measured electrophysiologically and anatomically (c-Fos) provide an index of regionally increased neuronal (synaptic) activity. We observed an increase in extracellular glutamate concentrations in the rVLM during BK-gallbladder stimulation but found no changes in glutamate concentrations sampled in areas outside the rVLM despite an increase in blood pressure during visceral reflex activation. These data suggest that rVLM neurons activated by BK-gallbladder stimulation rather than surrounding regions contribute to the increase in glutamate release. This conclusion reinforces our observation of the large number of glutamatergic rVLM neurons activated after gallbladder stimulation, identified by double-labeling with c-Fos and VGLUT3.
Chemical stimulation of the rVLM leads to release of glutamate in the IML, and sympathoexcitatory responses are mediated by NMDA receptors (55). NMDA and AMPA receptors, the two major ionotropic glutamate receptors in the rVLM, appear to mediate cardiovascular reflex responses to stimulation of somatic afferents (7, 32). We observed that blockade of either NMDA receptors with AP-5 or AMPA receptors with CNQX in the rVLM abolished the reflex pressor responses evoked by BK stimulation of visceral afferents, indicating that both NMDA and AMPA receptors also mediate the visceral reflex. These results supplement the observation that spinal NMDA and AMPA receptors mediate the reflex response to abdominal ischemia (21). The role of metabotropic glutamate receptors in the visceral reflex was not evaluated in the present study and remains a topic for future investigation.
Anesthetics such as ketamine can mask the contribution of spinal sensory NMDA and, to a lesser extent, spinal sensory AMPA receptors (1, 21). However, our results showed that blockade of each of the two ionotropic glutamate receptors in the rVLM similarly attenuated the visceral pressor reflex, indicating that even in the presence of ketamine, NMDA receptors in the brain stem functioned in the visceral cardiovascular reflex.
Glutamate regulates sympathetic activity in many brain areas concerned with processing cardiovascular reflexes, including the rVLM (40). Sympathetic premotor neurons originating in the rVLM project to the intermediolateral columns of the spinal cord and, in turn, regulate blood pressure and heart rate (3, 10, 59, 61). These rVLM premotor neurons contain glutamate as well as a variety of other excitatory neurotransmitters, including serotonin and catecholamines (44). Visceral reflex activation enhances the activity of sympathetic cardiovascular premotor neurons in the rVLM (58, 59, 61). The present study further elucidates the role of glutamate in regulating the sympathetic premotor neurons in the rVLM during visceral sympathoexcitatory reflexes by observing that iontophoresis of glutamate enhances the activity of the cardiovascular premotor neurons in the rVLM responsive to visceral afferent stimulation, whereas kynurenic acid, a glutamatergic ionotropic receptor antagonist, inhibits the action of exogenous glutamate as well as the excitatory response to stimulation of visceral afferents. These data strongly reinforce the notion that glutamate ionotropic receptors mediate activation of rVLM sympathetic premotor neurons during visceral cardiovascular reflex stimulation.
Although it seems clear that glutamate modulates excitatory responses of rVLM premotor sympathetic neurons, other excitatory (e.g., catecholamines, serotonin among others) and inhibitory neurotransmitters (e.g., opioids, nociceptin, and GABA) likely also play a role in processing neuronal activity in this brain stem region. Future studies are needed to evaluate interactions between these neurotransmitter systems to fully define the processing mechanisms in this sympathoregulatory region.
In summary, the present study provides novel evidence showing that glutamatergic neurons in the rVLM play a significant role in processing information during stimulation of visceral sympathetic/spinal afferent pathways. This excitatory cardiovascular visceral reflex requires medullary transmission through glutamatergic neurons functioning through stimulation of both NMDA and AMPA ionotropic receptors. In contrast to somatic reflexes, baroreflex activation is not required for visceral-rVLM glutamate release. Taken together, these data show that glutamate serves as an important excitatory neurotransmitter in brain stem processing of visceral sympathoexcitatory cardiovascular reflexes.
GRANTS
This study was supported by National Heart, Lung, and Blood Institute Grants HL-63313, HL-72125, and HL-66217 and the Larry K. Dodge Endowed Chair (to J. C. Longhurst). W. Zhou (Y. Syuu) is a recipient of a Research Fellowship Award from the American Heart Association-Western States Affiliate.
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.
The authors thank Natasha Tanya Balbas and Brenda Gongalez for excellent technical assistance with HPLC-electrochemical detection.
REFERENCES
- 1 Adreani CM, Hill JM, and Kaufman MP. Intrathecal blockade of both NMDA and non-NMDA receptors attenuates the exercise pressor reflex in cats. J Appl Physiol 80: 315–322, 1996.
Link | ISI | Google Scholar - 2 Ally A. Ventrolateral medullary control of cardiovascular activity during muscle contraction. Neurosci Biobehav Rev 23: 65–86, 1998.
Crossref | PubMed | ISI | Google Scholar - 3 Amendt K, Czachurski J, Dembowsky K, and Seller H. Bulbospinal projections to the intermediolateral cell column: a neuroanatomical study. J Auton Nerv Syst 1: 103–117, 1979.
Crossref | PubMed | Google Scholar - 4 Asmundsson G, Mokler DJ, and Ally A. Effects of ventrolateral medullary NMDA-receptor antagonism on biogenic amines and pressor response to muscle contraction. Neurosci Res 32: 47–56, 1998.
Crossref | PubMed | ISI | Google Scholar - 5 Baker DA, Xi ZX, Shen H, Swanson CJ, and Kalivas PW. The origin and neuronal function of in vivo nonsynaptic glutamate. J Neurosci 22: 9134–9141, 2002.
Crossref | PubMed | ISI | Google Scholar - 6 Barman SM and Gebber GL. Subgroups of rostral ventrolateral medullary and caudal medullary raphe neurons based on patterns of relationship to sympathetic nerve discharge and axonal projections. J Neurophysiol 77: 65–75, 1997.
Link | ISI | Google Scholar - 7 Bauer RM, Iwamoto GA, and Waldrop TG. Ventrolateral medullary neurons modulate pressor reflex to muscular contraction. Am J Physiol Regul Integr Comp Physiol 257: R1154–R1161, 1989.
Link | Google Scholar - 8 Berman AL. The Brainstem of Cats: A Cytoarchitectonic Atlas with Stereotaxic Coordinates. Madison, WI: The University of Wisconsin Press, 1968.
Google Scholar - 9 Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL, Pozzan T, and Volterra A. Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391: 281–285, 1998.
Crossref | PubMed | ISI | Google Scholar - 10 Brown DL and Guyenet PG. Cardiovascular neurons of brain stem with projections to spinal cord. Am J Physiol Regul Integr Comp Physiol 247: R1009–R1016, 1984.
Link | ISI | Google Scholar - 11 Chan RK and Sawchenko PE. Spatially and temporally differentiated patterns of c-fos expression in brainstem catecholaminergic cell groups induced by cardiovascular challenges in the rat. J Comp Neurol 348: 433–460, 1994.
Crossref | PubMed | ISI | Google Scholar - 12 Chao DM, Shen LL, Tjen-A-Looi S, Pitsillides KF, Li P, and Longhurst JC. Naloxone reverses inhibitory effect of electroacupuncture on sympathetic cardiovascular reflex responses. Am J Physiol Heart Circ Physiol 276: H2127–H2134, 1999.
Link | ISI | Google Scholar - 13 Collingridge GL and Lester RA. Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol Rev 41: 143–210, 1989.
PubMed | ISI | Google Scholar - 14 Crisostomo MM, Li P, Tjen-A-Looi SC, and Longhurst JC. Nociceptin in rVLM mediates electroacupuncture inhibition of cardiovascular reflex excitatory response in rats. J Appl Physiol 98: 2056–2063, 2005.
Link | ISI | Google Scholar - 15 Dampney RA. Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74: 323–364, 1994.
Link | ISI | Google Scholar - 16 Dampney RA and McAllen RM. Differential control of sympathetic fibres supplying hindlimb skin and muscle by subretrofacial neurons in the cat. J Physiol 395: 41–56, 1988.
Crossref | PubMed | ISI | Google Scholar - 17 Fonnum F. Glutamate: a neurotransmitter in mammalian brain. J Neurochem 42: 1–11, 1984.
Crossref | PubMed | ISI | Google Scholar - 18 Fremeau RT, Burman J, Qureshi T, Tran CH, Proctor J, Johnson J, Zhang H, Sulzer D, Copenhagen DR, Storm-Mathisen J, Reimer RJ, Chaudhry FA, and Edwards RH. The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate. Proc Natl Acad Sci USA 99: 14488–14493, 2002.
Crossref | PubMed | ISI | Google Scholar - 19 Fu LW, Zhou W, Guo ZL, and Longhurst JC. Acupuncture modulates release of glutamate in the rVLM during stimulation of visceral afferents. J Neuroscience 2004, p. 951.917.
ISI | Google Scholar - 20 Gatti PJ, Norman WP, Taveira Dasilva AM, and Gillis RA. Cardiorespiratory effects produced by microinjecting l-glutamic acid into medullary nuclei associated with the ventral surface of the feline medulla. Brain Res 381: 281–288, 1986.
Crossref | PubMed | ISI | Google Scholar - 21 Gee BY, Tjen-A-Looi SC, Hill JM, Chahal PS, and Longhurst JC. Role of spinal NMDA and non-NMDA receptors in the pressor reflex response to abdominal ischemia. Am J Physiol Regul Integr Comp Physiol 282: R850–R857, 2002.
Link | ISI | Google Scholar - 22 Guo ZL, Lai HC, and Longhurst JC. Medullary pathways involved in cardiac sympathoexcitatory reflexes in the cat. Brain Res 925: 55–66, 2002.
Crossref | PubMed | ISI | Google Scholar - 23 Guo ZL and Longhurst JC. Activation of nitric oxide-producing neurons in the brain stem during cardiac sympathoexcitatory reflexes in the cat. Neuroscience 116: 167–178, 2003.
Crossref | PubMed | ISI | Google Scholar - 24 Guo ZL, Moazzami AR, and Longhurst JC. Stimulation of cardiac sympathetic afferents activates glutamatergic neurons in the parabrachial nucleus: relation to neurons containing nNOS. Brain Res 1053: 97–107, 2005.
Crossref | PubMed | ISI | Google Scholar - 25 Guyenet P. Role of ventral medulla oblongata in blood pressure regulation. In: Central Regulation of Autonomic Functions, edited by Loewy AD and Spyer KM: New York: Oxford University Press, 1990, p. 145–167.
Google Scholar - 26 Guyenet PG, Haselton JR, and Sun MK. Sympathoexcitatory neurons of the rostral ventrolateral medulla and the origin of the sympathetic vasomotor tone. In: Progress in Brain Research, edited by Ciriello J, Caverson MM, and Polosa C: Amsterdam, The Netherlands, Elsevier, 1989, p. 105–115.
PubMed | ISI | Google Scholar - 27 Hand GA, Potts JT, Treuhaft BS, Wilson LB, Petty F, and Mitchell JH. Static muscle contraction elicits a baroreflex-dependent increase in glutamate concentration in the ventrolateral medulla. Brain Res 748: 211–218, 1997.
Crossref | PubMed | ISI | Google Scholar - 28 Ishide T, Maher TJ, Pearce WJ, Nauli SM, Chaiyakul P, and Ally A. Simultaneous glutamate and γ-aminobutyric acid release within ventrolateral medulla during skeletal muscle contraction in intact and barodenervated rats. Brain Res 923: 137–146, 2001.
Crossref | PubMed | ISI | Google Scholar - 29 Ishide T, Mancini M, Maher TJ, Chayaikul P, and Ally A. Rostral ventrolateral medulla opioid receptor activation modulates glutamate release and attenuates the exercise pressor reflex. Brain Res 865: 177–185, 2000.
Crossref | PubMed | ISI | Google Scholar - 30 Jacobs WA, Leburg MW, and Madaj EJ. Stability of o-phthalaldehyde-derived isoindoles. Anal Biochem 156: 334–340, 1986.
Crossref | PubMed | ISI | Google Scholar - 31 Katahira K, Mikami H, Otsuka A, Moriguchi A, Kohara K, Higashimori K, Okuda N, Nagano M, Morishita R, and Ogihara T. Differential control of vascular tone and heart rate by different amino acid neurotransmitters in the rostral ventrolateral medulla of the rat. Clin Exp Pharmacol Physiol 21: 545–556, 1994.
Crossref | PubMed | ISI | Google Scholar - 32 Kiely JM and Gordon FJ. Non-NMDA receptors in the rostral ventrolateral medulla mediate somatosympathetic pressor responses. J Auton Nerv Syst 43: 231–240, 1993.
Crossref | PubMed | Google Scholar - 33 Kobayashi T, Caringi D, Mokler DJ, and Ally A. Effects of ventrolateral medullary AMPA-receptor antagonism on pressor response during muscle contraction. Am J Physiol Heart Circ Physiol 272: H2774–H2781, 1997.
Link | ISI | Google Scholar - 34 Krukoff TL. c-Fos expression as a marker of functional activity in the brain: immunohistochemistry. In: Neuromethods, edited by Boulton AA, Baker GB, and Bateson AN. Totowa, NJ: Humana, 1998, p. 213–230.
Google Scholar - 35 Li P, Pitsillides KF, Rendig SV, Pan HL, and Longhurst JC. Reversal of reflex-induced myocardial ischemia by median nerve stimulation: a feline model of electroacupuncture. Circulation 97: 1186–1194, 1998.
Crossref | PubMed | ISI | Google Scholar - 36 Li P, Tjen-A-Looi S, and Longhurst JC. Rostral ventrolateral medullary opioid receptor subtypes in the inhibitory effect of electroacupuncture on reflex autonomic response in cats. Auton Neurosci 89: 38–47, 2001.
Crossref | PubMed | ISI | Google Scholar - 37 Lillaney R, Maher TJ, Chaiyakul P, and Ally A. Changes in extracellular glutamate and pressor response during muscle contraction following AMPA-receptor blockage in the RVLM and CVLM. Brain Res 844: 164–173, 1999.
Crossref | PubMed | ISI | Google Scholar - 38 Longhurst JC. Neural regulation of the cardiovascular system. In: Fundamental Neuroscience (2nd ed.), edited by Squire LR, Bloom FE, McConnell SK, Roberts JL, Spitzer NC, and Zigmond MJ. San Diego, CA: Academic Press, 2003, p. 935–966.
Google Scholar - 39 Longhurst JC, Spilker HL, and Ordway GA. Cardiovascular reflexes elicited by passive gastric distension in anesthetized cats. Am J Physiol Heart Circ Physiol 240: H539–H545, 1981.
Link | Google Scholar - 40 McCall RB. Role of neurotransmitters in the central regulation of the cardiovascular system. Prog Drug Res 35: 25–84, 1990.
PubMed | Google Scholar - 41 Meller R, Harrison PJ, Elliott JM, and Sharp T. In vitro evidence that 5-hydroxytryptamine increases efflux of glial glutamate via 5-HT(2A) receptor activation. J Neurosci Res 67: 399–405, 2002.
Crossref | PubMed | ISI | Google Scholar - 42 Miyawaki T, Minson J, Arnolda L, Llewellyn-Smith I, Chalmers J, and Pilowsky P. AMPA/kainate receptors mediate sympathetic chemoreceptor reflex in the rostral ventrolateral medulla. Brain Res 726: 64–68, 1996.
PubMed | ISI | Google Scholar - 43 Morimoto S, Sasaki S, Miki S, Kawa T, Nakamura K, Ichida T, Itoh H, Nakata T, Takeda K, Nakagawa M, and Yamada H. Pressor response to compression of the ventrolateral medulla mediated by glutamate receptors. Hypertension 33: 1207–1213, 1999.
Crossref | PubMed | ISI | Google Scholar - 44 Morrison SF, Callaway J, Milner TA, and Reis DJ. Rostral ventrolateral medulla: a source of the glutamatergic innervation of the sympathetic intermediolateral nucleus. Brain Res 562: 126–135, 1991.
Crossref | PubMed | ISI | Google Scholar - 45 Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, and Haydon PG. Glutamate-mediated astrocyte-neuron signaling. Nature 369: 744–747, 1994.
Crossref | PubMed | ISI | Google Scholar - 46 Pasti L, Zonta M, Pozzan T, Vicini S, and Carmignoto G. Cytosolic calcium oscillations in astrocytes may regulate exocytotic release of glutamate. J Neurosci 21: 477–484, 2001.
Crossref | PubMed | ISI | Google Scholar - 47 Philippu A. Regulation of blood pressure by central neurotransmitters and neuropeptides. Rev Physiol Biochem Pharmacol 111: 1–115, 1988.
Crossref | PubMed | ISI | Google Scholar - 48 Reidman DA, Maher TJ, Chaiyakul P, and Ally A. Modulation of extracellular glutamate and pressor response to muscle contraction during NMDA-receptor blockade in the rostral ventrolateral medulla. Neurosci Res 36: 147–156, 2000.
Crossref | PubMed | ISI | Google Scholar - 49 Ross CA, Ruggiero DA, Joh TH, Park DH, and Reis DJ. Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons. J Comp Neurol 228: 168–185, 1984.
Crossref | PubMed | ISI | Google Scholar - 50 Schafer MK, Varoqui H, Defamie N, Weihe E, and Erickson JD. Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons. J Biol Chem 277: 50734–50748, 2002.
Crossref | PubMed | ISI | Google Scholar - 51 Singewald N and Philippu A. Involvement of biogenic amines and amino acids in the central regulation of cardiovascular homeostasis. Trends Pharmacol Sci 17: 356–363, 1996.
Crossref | PubMed | ISI | Google Scholar - 52 Stornetta RL, Morrison SF, Ruggiero DA, and Reis DJ. Neurons of rostral ventrolateral medulla mediate somatic pressor reflex. Am J Physiol Regul Integr Comp Physiol 256: R448–R462, 1989.
Link | ISI | Google Scholar - 53 Sun MK and Guyenet PG. Medullospinal sympathoexcitatory neurons in normotensive and spontaneously hypertensive rats. Am J Physiol Regul Integr Comp Physiol 250: R910–R917, 1986.
Link | ISI | Google Scholar - 54 Sun MK, Hackett JT, and Guyenet PG. Sympathoexcitatory neurons of rostral ventrolateral medulla exhibit pacemaker properties in the presence of a glutamate-receptor antagonist. Brain Res 438: 23–40, 1988.
Crossref | PubMed | ISI | Google Scholar - 55 Sundaram K and Sapru H. NMDA receptors in the intermediolateral column of the spinal cord mediate sympathoexcitatory cardiac responses elicited from the ventrolateral medullary pressor area. Brain Res 544: 33–41, 1991.
Crossref | PubMed | ISI | Google Scholar - 56 Takamori S, Rhee JS, Rosenmund C, and Jahn R. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407: 189–194, 2000.
Crossref | PubMed | ISI | Google Scholar - 57 Timmerman W and Westerink BH. Brain microdialysis of GABA and glutamate: what does it signify? Synapse 27: 242–261, 1997.
Crossref | PubMed | ISI | Google Scholar - 58 Tjen-A-Looi SC, Li P, and Longhurst JC. Medullary substrate and differential cardiovascular responses during stimulation of specific acupoints. Am J Physiol Regul Integr Comp Physiol 287: R852–R862, 2004.
Link | ISI | Google Scholar - 59 Tjen-A-Looi SC, Li P, and Longhurst JC. Prolonged inhibition of rostral ventral lateral medullary premotor sympathetic neurons by electroacupuncture in cats. Auton Neurosci 106: 119–131, 2003.
Crossref | PubMed | ISI | Google Scholar - 60 Wu WC, Kuo JS, Wang Y, and Chai CY. Glycine increases arterial pressure and augments NMDA-induced pressor responses in the dorsomedial and ventrolateral medulla of cats. J Auton Nerv Syst 67: 145–155, 1997.
Crossref | PubMed | Google Scholar - 61 Zhou W, Tjen-A-Looi SC, and Longhurst JC. Brain stem mechanisms underlying acupuncture modality-related modulation of cardiovascular responses in rats. J Appl Physiol 99: 851–860, 2005.
Link | ISI | Google Scholar
