Differential Roles of NMDA and Non-NMDA Receptors in Synaptic Responses of Neurons in Nucleus Tractus Solitarii of the Rat

J. C. Yen,1 Julie Y. H. Chan,2 and Samuel H. H. Chan3

 1Institute of Pharmacology, National Yang-Ming University, Taipei 11221;  2Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Kaohsiung 81346; and  3Center for Neuroscience and Department of Biological Science, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, Republic of China


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Yen, J. C., Julie Y. H. Chan, and Samuel H. H. Chan. Differential roles of NMDA and non-NMDA receptors in synaptic responses of neurons in nucleus tractus solitarii of the rat. The relative role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in synaptic responses of neurons in caudal nucleus tractus solitarii (cNTS) was delineated by immunohistochemical and electrophysiologic experiments in rats. Double immunohistochemical staining in in vivo experiments revealed that ~80% of cNTS neurons that showed Fos-like immunoreactivity induced by baroreceptor activation were generally also immunoreactive to non-NMDA receptor subunits GluR1 or GluR2. On the other hand, only 20% of Fos-labeled cNTS neurons showed immunoreactivity to NMDA receptor subunits NMDAR1 or NMDAR2. Stimulation of the ipsilateral solitary tract at suprathreshold intensity in slice preparations induced Fos expression in the cNTS and evoked either a single action potential or a complex synaptic response consisting of an initial action potential followed by a secondary slow depolarization. In a majority (70%) of cNTS neurons that exhibited the complex synaptic response, both the initial and secondary components were eliminated reversibly by 6-cyano-7-nitroquinoxaline-2,3-dione (20 µM). This non-NMDA antagonist also inhibited the single action potential manifested by the other population of cNTS neurons. On the other hand, only the secondary slow depolarization was blocked by D(-)-2-amino-5-phosphonopentanoic acid (250 µM) or potentiated by NMDA (1.7 µM). Our results suggested that NMDA and non-NMDA receptors are involved differentially in the synaptic responses of cNTS neurons. Non-NMDA receptors may be distributed predominantly on a majority of the second-order cNTS neurons that may receive primary baroreceptor afferent inputs. On the other hand, NMDA receptors are located primarily on higher-order neurons, which may be connected reciprocally with the second-order cNTS neurons.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Considerable anatomic (Ciriello 1983; Housley et al. 1987; Kalia and Sullivan 1982) and electrophysiologic (Donoghue et al. 1984, 1985; Mifflin et al. 1988; Mifflin and Felder, 1990) observations indicate that, as the central termination site for baroreceptor afferent fibers (see Spyer 1981 for review), the caudal nucleus tractus solitarii (cNTS) plays a key role within the neural circuitry for baroreceptor reflex. In the search for the identity of neurotransmitters released at the cNTS after baroreceptor activation (Andresen and Kunze 1994; Lawrence and Jarrott 1996), one chemical substance that has been put forward is the excitatory amino acid, L-glutamate (Lawrence and Jarrott 1994; Ohta et al. 1996; Perrone 1981; Talman et al. 1980). However, controversy still exists regarding the role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the synaptic transmission of primary visceral afferents at the cNTS. For example, pharmacological studies variably demonstrated the participation of predominantly NMDA (Kubo and Kihara 1988) or non-NMDA (Gordon and Leone 1991) receptors or both types of ionotropic glutamatergic receptors (Kubo and Kihara 1991; Ohta and Talman 1994) in the synaptic processes engaged by the baroreceptor afferents at the cNTS. Electrophysiologic data similarly suggest that synaptic responses of cNTS neurons to solitary tract stimulation are either mediated primarily by non-NMDA receptors (Andresen and Yang 1990) or by both NMDA and non-NMDA receptors (Aylwin et al. 1997; Brooks and Spyer 1993; Miller and Felder 1988; Titz and Keller 1997).

Immunohistochemical demonstration of Fos, the nuclear protein encoded by the proto-oncogene c-fos, has been documented in recent years to be a powerful tool for tracing synaptically activated neurons in the CNS (Dampney et al. 1995). Fos-like immunoreactivity (Fos-LI) is detected in rat cNTS neurons after hypertension (Graham et al. 1995; Miura et al. 1994; Murphy et al. 1994; Shih et al. 1996), electrical stimulation of the carotid sinus nerve (Erickson and Millhorn 1991) or aortic depressor nerve (McKitrick et al. 1992), or increase in carotid sinus pressure (Dean and Seagard 1995). We demonstrated in a recent study (Chan et al. 1998) that both NMDA and non-NMDA receptors are involved in Fos expression by cNTS neurons in response to baroreceptor activation. One intriguing finding, which formed the immediate impetus for the present study, is that double immunohistochemical staining revealed that cNTS neurons that manifest Fos-LI in response to transient hypertension are generally also immunoreactive to GluR1 subunit of non-NMDA receptors. On the other hand, Fos expression is usually absent from cNTS neurons that are immunoreactive to NMDA receptor subunit NMDAR1.

Arising from the foregoing discussion are at least two, perhaps not mutually exclusive, possibilities on the role of NMDA and non-NMDA receptors in the synaptic processes engaged by primary afferents at the cNTS. First, NMDA and non-NMDA receptors are colocalized on second-order cNTS neurons that receive glutamatergic sensory afferent inputs (Fig. 1A). Second, whereas non-NMDA receptors are distributed predominantly on second-order cNTS neurons, NMDA receptors are located primarily on higher-order neurons that are reciprocally connected with second-order neurons (Fig. 1B). These two possibilities were evaluated in the present study based on immunohistochemical and electrophysiologic experiments.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1. Schematic drawing showing 2 possibilities on the role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in the synaptic processes engaged by baroreceptor afferents at the caudal nucleus tractus solitarii (cNTS). A: NMDA and non-NMDA receptors may colocalize on second-order neurons that receive glutamatergic primary afferent inputs. B: non-NMDA receptors and NMDA receptors may be located on second- and higher-order cNTS neuron, respectively, in the baroreceptor reflex loop, and that these cNTS neurons may be interconnected reciprocally.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

The procedures used in both our in vivo and in vitro experiments were approved by the Experimental Animal Committee of the National Yang-Ming University. Male, adult Sprague-Dawley rats (250-300 g) used in in vivo study were anesthetized initially with pentobarbital sodium (50 mg/kg ip). Routine preparatory surgery included intubation of the trachea to facilitate ventilation and cannulation of the right femoral artery and vein to measure systemic arterial pressure and administer drugs. To provide satisfactory anesthetic maintenance (Yang et al. 1996), the left femoral vein also was cannulated for continuous infusion of pentobarbital sodium (10-20 mg · kg-1 · h-1 iv)

In vivo experiments

INDUCTION OF FOS-LIKE PROTEIN BY BARORECEPTOR ACTIVATION. As described previously (Chan et al. 1998), we induced Fos expression in cNTS neurons in in vivo experiments by repeated and scheduled activation of the baroreceptors. In essence, during a period of 120 min, transient hypertension evoked by three separate doses of phenylephrine (2.5, 5, or 10 µg/kg iv) was executed at 0-10, 10-20, and 110-120 min. The order of different doses of phenylephrine, which was administered within 10 min, was altered randomly to avoid sequential dependency on changes in systemic arterial pressure. The injection volume was restricted to 250 µl to reduce alterations in blood volume. Animals that received repeated and scheduled intravenous administration of saline served as the vehicle control. Rats that were subjected to surgical preparation without further experimental procedures served as the sham control.

DOUBLE IMMUNOHISTOCHEMICAL STAINING FOR FOS-LIKE PROTEIN AND GLUTAMATE RECEPTOR SUBTYPES. At the end of the 120 min of repeated and scheduled baroreceptor activation, the brain stem of the animal was processed for double immunohistochemical staining for Fos-like protein and glutamate receptor subtypes, as described in our recent studies (Chan et al. 1997, 1998; Shih et al. 1996). In brief, frozen serial transverse sections (20 µm) of the medulla oblongata were cut on a cryostat (Leica CM3050). They first were processed for Fos-LI, using a polyclonal sheep anti-Fos antiserum (1:4000; Cambridge Research Biochemical). Immunoreactivity was visualized by the avidin-biotin peroxidase complex (ABC) method with nickel intensification (Vector ABC-Elite Kit; Vector Laboratories). The same sections subsequently were incubated with a polyclonal rabbit antiserum (Chemicon) directed against either the C-terminus peptide of rat AMPA-type non-NMDA receptor subunit 1 (GluR1; 1:1000) or subunit 2 (GluR2; 1:1000) or the C-terminus peptide of rat NMDA receptor subunit 1 (NMDAR1; 1:2000) or subunit 2 (NMDAR2; 1:2000). Immunoreactive product again was visualized by the Vector ABC-Elite Kit, staining for alkaline phosphatase. The final immunohistochemical product of Fos-LI was stained in black and that of GluR1, GluR2, NMDAR1 or NMDAR2 was stained in dark red.

As described previously (Chan et al. 1998), the specificity of our double immunohistochemical staining was confirmed by purposely mismatching the secondary antiserum. Specifically, incubation with sheep anti-Fos antiserum was followed by biotinylated anti-rabbit IgG or biotinylated anti-sheep IgG was used after rabbit anti-NMDAR1, anti-NMDAR2, anti-GluR1 or anti-GluR2 antiserum. Sections then were reacted together with the experimental tissues. No specific immunoreactivity was observed in these control sections. In some experiments, the order of immunostaining for Fos protein and glutamate receptor subunits was reversed to confirm the validity of the immunoreactivity detected by double-labeling procedures. In other control experiments, sections were incubated in the absence of either the primary antiserum or the antiserum was substituted with either normal sheep serum (Fos antiserum) or normal rabbit serum (glutamate antiserum). Again no specific immunoreactivity was observed in these control sections when they were processed together with the experimental tissues.

QUANTIFICATION OF DOUBLE IMMUNOHISTOCHEMICAL REACTION PRODUCTS IN THE CNTS. Tissue sections were examined under bright-field microscopy to quantify the relationship between Fos-LI and immunoreactivity for GluR1, GluR2, NMDAR1, or NMDAR2 subunit in the cNTS (Chan et al. 1998). The criterion for identification of Fos-immunoreactive neurons was a distinctly stained nucleus (Chan et al. 1997, 1998; Murphy et al. 1994; Shih et al. 1996). To qualify as double-labeled cNTS neurons, the area occupied by the immunohistochemical stain for individual glutamate receptor subunits must at least equal that of the Fos-positive nucleus.

The caudal medulla oblongata was divided into eight levels, at 200-µm intervals, between 1.2 mm caudal and 0.2 mm rostral to the obex. Five sections selected randomly from each level were counted separately by two individuals, and the mean number of the Fos-positive cells for each level of the NTS was tabulated. Double counting was corrected using the method suggested by Abercrombie (1946). Photomicrographs were taken with a Nikon inverted microscope equipped with Nomarski optics or a Leitz microscope.

In vitro experiments

SLICE PREPARATION. The procedures used in in vitro experiments were described in detail in our previous study (Yen and Chan 1997). Briefly, transverse slices (350-400 µm) of the medulla oblongata, at the level of obex, were obtained from young male Sprague-Dawley rats (150-200 g) that were decapitated with a guillotine. Slices were transferred immediately to well-oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) and were allowed to be stabilized for >= 1 h before commencement of the experiment. The composition of the ACSF was (in mM) 117 NaCl, 4.7 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 25 NaHCO3, and 11 glucose.

ELECTROPHYSIOLOGIC RECORDING. During the experiment, the slice was placed on a nylon mesh in an interface-type brain slice chamber and was perfused with well-oxygenated (95% O2-5% CO2), warm (32 ± 2°C; mean ± SE) ACSF at a flow rate of 2-4 ml/min. Glass micropipettes (WPI) filled with 4 M potassium acetate solution (impedance: 50-100 MOmega ) were used for intracellular recording. The microelectrode was positioned under visual guidance with the aid of a dissection microscope. Recording sites were restricted principally within the dorsomedial area of the cNTS, the primary termination site for baroreceptor afferents (Ciriello 1983). The ipsilateral solitary tract was activated electrically to evoke synaptic responses of cNTS neurons to primary afferents. Constant square-wave current pulses (0.1 Hz, 0.1 ms, 40-400 µA) were delivered by a bipolar tungsten electrode (Rhodes Medical SNE-200; 0.1-mm contact diameter, 0.25-mm separation) via a photoelectric stimulus isolation unit (Grass PSIU6). The distance from stimulus site to recording electrode ranged 0.1-0.2 mm. We ascertained that the recorded cNTS neurons were second-order neurons according to the criteria suggested previously (Aylwin et al. 1997; Miles 1986) for monosynaptically evoked synaptic responses based on short and invariant onset latency and the ability to respond to paired stimuli at 5-ms intervals.

INDUCTION OF FOS EXPRESSION BY SOLITARY TRACT STIMULATION. Immunohistochemical staining for Fos-LI was carried out in some experiments to evaluate whether Fos expression can be induced in the cNTS by solitary tract stimulation in the slice preparation. For this purpose, the solitary tract was electrically activated for 120 min, using the same stimulus parameters as in our electrophysiologic experiments. The slice was thereafter fixed, sectioned and processed for Fos-LI as described in the preceding text, using the ABC method with nickel intensification.

Drugs

NMDA, D(-)-2-amino-5-phosphonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Research Biochemicals International. They were prepared freshly with ACSF immediately before use during the experiment and were applied by superfusion.

Statistics

All values are presented in means ± SE. Paired t-test was used, whenever applicable, to analyze the significance of drug treatments.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Differential localization of Fos-LI and immunoreactivity of NMDA or non-NMDA receptor subunits in cNTS

Figure 2 illustrates typical examples of double immunohistochemical staining for Fos-LI and glutamate receptor subunits. Similar to our recent finding (Chan et al. 1998; Shih et al. 1996), repeated and scheduled stimulation of baroreceptors by transient hypertension induced Fos expression in the cNTS. A majority of the Fos-immunoreactive cNTS neurons, which manifested a distinctively stained nucleus (Fig. 2), was present in the commissural, medial, and dorsomedial subnuclei. Neurons in the same subnuclei of the cNTS also were immunostained heavily for GluR1 (Fig. 2A), GluR2 (Fig. 2B), NMDAR1 (Fig. 2C), or NMDAR2 (Fig. 2D) subunit. These immunoreactive materials were present in the perikaryal cytoplasm, but not in the nucleus. Similar to previous studies (Chan et al. 1997, 1998; Shih et al. 1996), only basal Fos-LI was detected at the cNTS after repeated and scheduled intravenous administration of saline or in sham-control animals.



View larger version (194K):
[in this window]
[in a new window]
 
Fig. 2. Representative photomicrographs showing the relationship between Fos-like immunoreactivity (Fos-LI) at the caudal NTS in response to transient hypertension and immunoreactive product of GluR1 (A), GluR2 (B), NMDAR1 (C), or NMDAR2 (D). Small arrows denote cNTS neurons that were immunoreactive to both Fos and GluR1, GluR2, NMDAR1, or NMDAR2; large arrow heads denote cNTS neurons that were immunostained with only 1 of the 4 glutamatergic receptor subunits; small arrow heads denote neurons that showed only Fos-LI. Scale bar = 25 µm. ts, tractus solitarii.

Intriguingly, at all rostrocaudal levels of caudal NTS we examined, ~78.5% of cNTS neurons that showed Fos-LI were double labeled for GluR1 subunit (Figs. 2A and 3A). Comparable proportion (79.4%) of Fos-positive neurons was also immunoreactive to GluR2 subunit (Figs. 2B and 3B). On the other hand, much less Fos-labeled cNTS neurons showed immunoreactivity to either NMDAR1 (23.8%; Figs. 2C and 3C) or NMDAR2 (19.8%; Figs. 2D and 3D) subunit. We obtained comparable results from experiments in which the order of immunostaining for Fos protein and glutamate receptor subunits was reversed, ascertaining that these observations were not due to inadvertent reduction in the expression of the glutamate receptor subtypes because of the double-labeling procedures.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3. Distribution of neurons at 8 rostrocaudal levels of the cNTS with reference to the obex that showed only Fos-LI in response to transient hypertension () or were additionally immunoreactive () to GluR1 (A), GluR2 (B), NMDAR1 (C), or NMDAR2 (D). Values are presented as means ± SE, n = 4-5 animals per group.

Synaptic responses of cNTS neurons to electrical stimulation of the solitary tract

The primary aim of our electrophysiologic experiments was to evaluate the synaptic responses of second-order cNTS neurons to monosynaptic activation by primary afferent volleys in our slice preparation. Thus only cNTS neurons that exhibited a postsynaptic potential with short (<4.5 ms) and invariant (variability <0.5 ms) onset latency (Fig. 4, inset) and were able to follow paired solitary tract stimuli separated by 5 ms (Aylwin et al. 1997; Miles 1986) were included in our analysis. Of 55 cNTS neurons evaluated, 42 neurons showed a subthreshold response of excitatory postsynaptic potential (EPSP), with an onset latency of 2.9 ± 0.4 ms and a time-to-peak interval of 8.0 ± 2.5 ms. An action potential can be evoked in 39 of those 42 neurons with an onset latency of 2.9 ± 0.4 ms and a time-to-peak interval of 7.4 ± 2.6 ms when the solitary tract was stimulated at the rheobase stimulus intensity for each neuron. Twenty of these 39 cNTS neurons additionally manifested a slow depolarization of 6.8 ± 1.9 mV with a time-to-peak interval of 75.6 ± 16.8 ms and a duration of 247.3 ± 24.7 ms.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4. Illustrated example of synaptic responses of a cNTS neuron to electrical stimulation of the ipsilateral solitary tract (0.1 Hz, 0.1 ms, black-triangle) at 40 (Aa), 50 (Ab), 100 (Ac), 200 (Ad), or 400 (Ae) µA. Note that the EPSP evoked at a subthreshold intensity (Aa) gave way to an initial action potential, followed by a secondary slow depolarization on stimulation with suprathreshold intensities (A, b-e). Note also the appearance of multiple phasic EPSPs (Ad) or superimposed action potentials (Ae) at the higher intensities. B: responses depicted in A, a and c, were superimposed to show that the 2nd slow depolarization was generated only in the presence of the initial action potential. Resting transmembrane potential was -65 mV for this neuron. · · · , -70 mV. Inset: representative example of 10 consecutive synaptic responses with short and invariant onset latency in another cNTS neuron to electrical stimulation (0.1 Hz, 0.1 ms, 100 µA; black-triangle). down-arrow , point of initiation for the action potential.

Figure 4A illustrates the synaptic responses typical of the 20 cNTS neurons that exhibited a combination of an initial action potential and a secondary slow depolarization on stimulation of the solitary tract. The EPSP evoked at a subthreshold intensity of 40 µA (Fig. 4Aa) gave way to an action potential, followed by a prolonged depolarization (Fig. 4Ab), when the stimulus intensity was increased to 50 µA. Further increments in stimulus intensity (100, 200, or 400 µA) progressively shortened the time-to-peak interval (from 5.6 to 3.5 ms) for the initial action potential, without affecting discernibly the onset latency. At the same time, the secondary slow depolarization (Fig. 4A, c-e) underwent an increase in the amplitude (from 8.6 to 22.8 mV) or duration (from 250 to 364 ms) and a decrease in the time-to-peak interval (from 55.6 to 24.3 ms). We also noted that multiple phasic EPSPs (Fig. 4A, d and e), which may become action potentials (Fig. 4Ae), superimposed on the slow depolarization when the solitary tract was stimulated at higher stimulus intensities.

The above relationship between the synaptic responses of cNTS neurons and the intensity of stimulation of the solitary tract suggest the presence of a causative relationship between the initial action potential and the secondary slow depolarization. This suggestion was illustrated further when Fig. 4A, a and c, were superimposed (Fig. 4B). Whereas the EPSP evoked by a subthreshold stimulus exhibited a time-to-peak interval (5.9 ± 1.2 ms, n = 20) that was comparable with that of the initial action potential (5.6 ± 0.9 ms, n = 20), the slow depolarization was generated only in the presence of the initial action potential.

Effects of AP5 or CNQX on the initial action potential and secondary slow depolarization evoked by the solitary tract on cNTS neurons

We next used specific glutamate receptor antagonists to delineate the contribution of NMDA and non-NMDA receptors to the synaptic mechanisms that may underlie the elicitation of the initial action potential and secondary slow depolarization in cNTS neurons by stimulation of the solitary tract. In 14 of the 20 cNTS neurons that manifested the complex synaptic response, superfusion with AP5 (250 µM), a specific NMDA receptor antagonist, significantly (P < 0.05) abolished the secondary slow depolarization (from 6.3 ± 0.8 mV to 0.2 ± 0.1 mV, n = 14) without discernibly affecting the initial action potential and resting membrane potential (Fig. 5Ab). The slow depolarization subsequently recovered (6.4 ± 0.6 mV, n = 14) after superfusion with ACSF (Fig. 5Ac). Intriguingly, in addition to inhibiting the initial action potential, administration of the specific non-NMDA receptor antagonist, CNQX (20 µM), also eliminated the secondary slow depolarization (Fig. 5Ad). There was instead a minor hyperpolarization (-2.5 ± 0.6 mV, n = 14) that was sensitive to bicuculline (data not shown). Again, these synaptic events took place without apparent changes in resting membrane potential and were reversible (Fig. 5Ae). It should be mentioned that the singular action potential evoked in 19 cNTS also was eliminated reversibly by superfusion of the slice with CNQX (20 µM). These results suggest that the initial action potential evoked in these cNTS neurons by the solitary tract is mediated by non-NMDA receptors. Furthermore whereas NMDA receptors are responsible for the secondary slow depolarization, these receptors are likely to be present in higher-order cNTS neurons that are connected reciprocally with the second-order neurons.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5. A: illustrated example of synaptic responses of a cNTS neuron to electrical stimulation of the ipsilateral solitary tract (0.1 Hz, 0.1 ms, black-triangle) at 100 µA, before (a) and during superfusing the brain slice with D(-)-2-amino-5-phosphonopentanoic acid (AP5; 250 µM, b) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM; d). Note elimination of the secondary slow depolarization by AP5 (b), and inhibition of both the initial action potential and the prolonged depolarization by CNQX (d). Superfusion of the slice with artificial cerebrospinal fluid (ACSF, c and e) between drug application essentially restored the typical synaptic response. Resting transmembrane potential was -60 mV for this neuron. · · · , -70 mV. B: superimposed tracings illustrating the effect of AP5 (250 µM) on synaptic responses to subthreshold stimulation of the solitary tract (0.1 Hz, 0.1 ms, 40 µA; black-triangle) of the same neuron in A at its resting membrane potential of -60 mV (a) or when depolarized to -30 mV (b).

We are aware that at resting membrane potential, NMDA receptor currents are under a voltage-dependent blockade by Mg2+ ions (Aylwin et al. 1997; Titz and Keller 1997). Thus it is possible that this blockade may be associated with the seemingly lack of involvement of NMDA receptors in the initial action potential evoked in the second-order cNTS neurons. This possibility was addressed by examining the effect of AP5 on synaptic responses to solitary tract activation of cNTS neurons that were maintained under different depolarized states through intracellular current injection. As illustrated in Fig. 5B, the amplitude of EPSP evoked by subthreshold solitary tract stimulation was not significantly altered (13.4 ± 1.9 mV vs. 13.3 ± 1.7 mV, P > 0.05, n = 14) by AP5 (250 µM) when the membrane potential was maintained at -60 mV (Fig. 5Ba). With the exception of a smaller EPSP, comparable findings (4.6 ± 0.8 mV vs. 4.4 ± 0.9 mV, P > 0.05, n = 14) were obtained when the membrane potential was depolarized to -30 mV (Fig. 5Bb). At this membrane potential, the conductance gated by NMDA receptor channels is already half-maximally activated (Hestrin et al. 1990). Hence it is unlikely that the participation of NMDA-receptor-mediated response was masked by the Mg2+ blockade of these receptors in these cNTS neurons.

The remaining 6 of the 20 cNTS neurons manifested a different response pattern to AP5 and CNQX, as exemplified by Fig. 6. In this particular instance, electrical stimulation of the solitary tract at an intensity of 200 µA elicited two initial action potentials with a maintained interspike interval of 7.5 ms, to be followed by a third spike potential that occurred 30 ms later (Fig. 6, A, C, and F). Interestingly, superfusion with AP5 (250 µM) reversibly inhibited both the second and third action potentials (Fig. 6, B and C). On the other hand, whereas the first action potential was eliminated by CNQX (20 µM), the interval between the second and third action potentials was prolonged from 24.7 to 45.9 ms (Fig. 6D). Coadministration of both AP5 and CNQX removed all three evoked action potentials (Fig. 6E), and the blockade was reversed after superfusion with ACSF (Fig. 6F). These results suggest that both NMDA and non-NMDA receptors are present in these cNTS neurons and mediate respectively a slow and fast action potential (Wu and Kelly 1996) on stimulation of the solitary tract. In addition, these second-order cNTS neurons also may receive excitatory synaptic inputs from higher-order cNTS neurons where NMDA receptors are present.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 6. Illustrated example of synaptic responses of a cNTS neuron to electrical stimulation of the ipsilateral solitary tract (0.1 Hz, 0.1 ms, black-triangle) at 200 µA before (A) and during superfusing the brain slice with AP5 (250 µM, B), CNQX (20 µM; D), or both AP5 and CNQX (250 and 20 µM; E). Note elimination of the 2nd and 3rd action potentials by AP5 (B), inhibition of the 1st action potential and prolongation of the interval between the 2nd and 3rd action potentials by CNQX (D), and abolition of all evoked action potentials in the presence of both AP5 and CNQX (E). Superfusion of the slice with ACSF (C and F) between drug application essentially restored the typical synaptic response. Resting transmembrane potential was -62 mV for this neuron. · · · , -70 mV.

Additive effect of NMDA on the secondary slow depolarization evoked in cNTS neurons by stimulation of the solitary tract

To further ascertain the role of NMDA receptors in the elicitation of the secondary slow depolarization in a majority of cNTS neurons by afferent volleys from the solitary tract, we evaluated the effect of NMDA on this synaptic event. We noted in our previous study (Yen and Chan 1997) that superfusing the brain slice with NMDA at 6.8 µM increases the regular spontaneous discharges in cNTS neurons. To avoid this confounding factor, we have chosen to apply lower concentration of NMDA in this series of experiments. We found in all six cNTS neurons evaluated (Fig. 7, a and b) that superfusion of NMDA (1.7 µM) induced minimal effect on the initial EPSP evoked by subthreshold stimulation of the solitary tract (6.6 ± 1.2 mV vs. 6.8 ± 1.0 mV, P > 0.05, n = 6) and failed to elicit a secondary slow depolarization. However, the same drug treatment significantly (P < 0.05) enhanced the amplitude (from 6.3 ± 1.5 mV to 12.7 ± 1.8 mV, n = 6) and prolonged the duration (from 237.5 ± 18.2 ms to 298.7 ± 21.6 ms, n = 6) of the slow depolarization evoked by suprathreshold stimulation of the solitary tract (Fig. 7, c and d). The onset latency and time-to-peak of the initial action potential, on the other hand, was unaffected (Fig. 7, c and d). These results supported the observation that the slow excitation of cNTS neurons is a synaptically evoked consequence and is mediated specifically by NMDA receptors.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 7. Illustrated example of synaptic responses of a cNTS neuron to electrical stimulation of the ipsilateral solitary tract (0.1 Hz, 0.1 ms, black-triangle) at 50 µA (a and b) or 100 µA (c and d), before (a and c) and during (b and d) superfusing the brain slice with NMDA at 1.7 µM. Note enhanced amplitude and prolonged duration of the secondary slow depolarization only in d but not in b, without concurrent alterations in the initial synaptic responses. Resting transmembrane potential was -59 mV for this neuron. · · · , -70 mV.

Induction of Fos expression by electrical stimulation of the solitary tract in the slice preparation

We are concerned that the solitary tract contains nerve fibers that relay viscerosensory information other than baroreceptor signals (Loewy 1990). Thus erroneous interpretations may arise from correlating Fos expression in the cNTS induced by baroreceptor activation in our in vivo experiments with electrophysiologic response of cNTS neurons to solitary tract stimulation in our in vitro slice preparation. Figure 8 minimizes this concern by showing that, similar to baroreceptor activation in in vivo experiments, Fos-LI was detected in the cNTS after the solitary tract in our brain stem slice was activated electrically using the same stimulus parameters as in our electrophysiologic experiments. Furthermore a majority of these Fos-immunoreactive neurons was present in the commissural, medial and dorsomedial subnuclei of the cNTS.



View larger version (202K):
[in this window]
[in a new window]
 
Fig. 8. Representative photomicrograph showing the presence of Fos-LI at the caudal NTS in response to electrical stimulation of the solitary tract in the slice preparation. Scale bar = 25 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Based on corroborated immunohistochemical and electrophysiologic results, we demonstrated in the present study that non-NMDA receptors are predominantly distributed on a majority of the second-order cNTS neurons that receive primary baroreceptor afferent inputs. On the other hand, NMDA receptors are located primarily on the higher-order neurons, which are connected reciprocally with the second-order cNTS neurons in the baroreceptor reflex loop (Fig. 1B). We found in addition that both NMDA and non-NMDA receptors colocalize on a minority of the second-order cNTS neurons (Fig. 1A). Together these demonstrations resolved the controversy over the role of NMDA and non-NMDA receptors in the synaptic processes engaged by the primary visceral afferents at the cNTS.

The present results extended our previous findings (Chan et al. 1998) and provided the first immunohistochemical demonstration that both NMDA and AMPA-type non-NMDA receptors exist heterologously in rat cNTS neurons, containing respectively, NMDAR1/NMDAR2 and GluR1/GluR2 subunits. Several lines of evidence suggest that native NMDA and AMPA-type non-NMDA receptors are likely to comprise at least, respectively, NMDAR1/NMDAR2 and GluR1/GluR2 subunits (Luo et al. 1997; Sato et al. 1993; Sheng et al. 1994; Sucher et al. 1996). Molecular characterization of glutamate receptor subunits showed that NMDAR1 and NMDAR2 subunits are critical for the functional expression of NMDA receptors (Ishii et al. 1993; Monyer et al. 1992). Immunoprecipitation assays also indicated that NMDAR1 and NMDAR2 subunits coexist in native NMDA receptor of rat cortical neurons (Luo et al. 1997; Sheng et al. 1994; Sucher et al. 1996). GluR1 and GluR2 subunits play important structural and functional roles in non-NMDA receptor ion channels that are gated by AMPA (Boulter et al. 1990; Hollmann et al. 1991). In situ hybridization study further revealed that GluR1 and GluR2 subunit mRNAs are present predominantly in rat NTS neurons (Sato et al. 1993).

We found that whereas ~79% of cNTS neurons that showed Fos-LI in response to baroreceptor activation also were double-labeled for GluR1 or GluR2 subunit, 20-24% of Fos-labeled cNTS neurons showed immunoreactivity to either NMDAR1 or NMDAR2 subunit. By showing such a differential relationship with induced Fos expression in cNTS neurons, our present results suggest a novel functional role for NMDAR1/NMDAR2 and GluR1/GluR2 subunits in the neural circuitry that subserves the synaptic processes of baroreceptor afferents at the cNTS. This suggestion subsequently was validated by our electrophysiologic data. We found that both the initial action potential and secondary slow depolarization evoked by stimulation of the solitary tract in 70% of the cNTS neurons were inhibited by CNQX, whereas only the slow depolarization was eliminated by AP5. We further validated this suggestion by demonstrating (cf. Fig. 4B) that the secondary slow depolarization was generated only when stimulation of the solitary tract resulted in the initial action potential. On the other hand, this causative relationship failed to materialize on subthreshold solitary tract activation that evoked only an EPSP in the recorded cNTS neurons.

These observations support the notion that a majority of the second-order cNTS neurons are subject to dual excitation on the arrival of primary afferent volleys. Action potentials evoked initially in these neurons via primary afferent transmission and mediated by AMPA-type non-NMDA receptors subsequently may activate NMDA receptors on higher-order neurons. The latter neurons, in turn, may exert a secondary depolarization in second-order cNTS neurons by way of reciprocal innervation. Previous electrophysiologic and functional studies also suggest that NMDA receptors may be present predominantly on interneurons within the NTS but are relatively absent from neurons receiving primary afferent inputs (Andresen and Kunze 1994; Brooks and Spyer 1993). Zhang and Mifflin (1997, 1998) also showed recently that non-NMDA receptor subtypes play a major role in the transmission across the first synapse from baroreceptor afferents to second-order NTS neurons. On the other hand, both NMDA and non-NMDA receptors are involved in the integration of baroreceptor inputs by higher-order NTS neurons. Apart from the differential blunting effects of CNQX and AP5, we also confirmed that the relatively small contribution of NMDA receptors to the initial action potential evoked by solitary tract was not associated with the voltage-dependent Mg2+ blockade of NMDA receptor channels.

The validity of the aforementioned notion is contingent on the complementarity of Fos expression in the cNTS induced by baroreceptor activation in our in vivo experiments with electrophysiologic response of cNTS neurons to solitary tract stimulation in our in vitro preparation. In this regard, we demonstrated that under the same stimulus parameters as in our electrophysiologic experiments, electrical activation of the solitary tract in our slice preparation also resulted in Fos-LI in the cNTS. Furthermore the distribution of these Fos-immunoreactive neurons in subnuclei of the cNTS was comparable with that evoked by baroreceptor activation in in vivo experiments.

An important assumption in the present study was the cNTS neurons that showed Fos-LI immunoreactivity in our in vivo experiments, and the neurons that we evaluated electrophysiologically in our slice preparations were second-order cNTS neurons in the baroreceptor reflex loop. Primary afferent fibers from arterial baroreceptors terminate mainly in the commissural, medial, and dorsomedial subnuclei of the intermediate and caudal NTS (Ciriello 1983; Kalia and Sullivan 1982). It is of interest that Fos-positive cells that we identified in the cNTS in response to baroreceptor activation also distributed primarily in these subnuclei. As we pointed out previously (Chan et al. 1998; Shih et al. 1996), such a closely matched topographic distribution of baroreceptor afferent termination sites and Fos-positive neurons suggests that these Fos-labeled cNTS neurons represent second-order neurons in the baroreceptor reflex pathways. Similar conclusion was drawn recently by Chan and Sawchenko (1998). To conform with these anatomic features, the neurons we recorded were restricted to the dorsomedial subnuclei of the cNTS. In addition, only neurons that satisfied the criteria for a monosynaptically activated neuron (Aylwin et al. 1997; Miles 1986) were evaluated in our electrophysiologic experiments. The short onset latency (2.9 ± 0.4 ms) and time-to-peak interval (5.6 ± 0.9 ms) of the initial action potentials evoked in the cNTS neurons are within the range reported (Andresen and Yang 1990; Miller and Felder 1988) for second-order cNTS neurons to solitary tract activation.

We are cognizant that a prolonged depolarization was reported in NTS neurons in response to electrical stimulation of either the peripheral afferent fibers in in vivo experiments (Mifflin and Felder 1988) or solitary tract in in vitro preparations (Champagnat et al. 1986; Fortin and Champagnat 1993; Kawai and Senba 1996; Miles 1986; Paton et al. 1993). A re-excitatory mechanism has been proposed to account for this slow synaptic response (Fortin and Champagnat 1993; Kawai and Senba 1996; Paton et al. 1993). Under this proposed mechanism, synaptically evoked multiple EPSPs during the slow excitatory component, as we also observed on stimulation of the solitary tract with higher stimulus intensities (cf. Fig. 4), are considered to be a characteristics of re-excitatory processing (Fortin and Champagnat 1993). Mayer and Westbrook (1987) also proposed a "recurrent excitation" model to explain the long duration of responses to synaptic activation of NMDA receptors in vertebrate central neurons. The contribution of our immunohistochemical and electrophysiologic results against this information resides in defining the neuronal circuit and sequence of synaptic events at the cNTS that lead to a NMDA receptor-mediated re-excitatory mechanism that contributes to the slow excitation in response to solitary tract activation. Our pharmacological experiments with superfusion of low-dose NMDA (cf. Fig. 7) provided further credence to this notion.

In conclusion, our immunohistochemical and electrophysiologic results support the notion that non-NMDA receptors may be predominantly distributed on second-order cNTS neurons and transmit primary afferent information. NMDA receptors, on the other hand, are located mainly on higher-order neurons that form a re-excitatory network with second-order neurons and may physiologically play a modulatory role in baroreceptor reflex.


    ACKNOWLEDGMENTS

This study was carried out during S.H.H. Chan's tenure as National Chair Professor in Neurosciene as appointed by the Ministry of Education, and was supported in part by research grant NSC87-2314-B010-037-M10 to S.H.H. Chan from the National Science Council, Taiwan, Republic of China.


    FOOTNOTES

Address reprint requests to S.H.H. Chan.

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.

Received 6 July 1998; accepted in final form 2 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society