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Am J Physiol Regul Integr Comp Physiol 278: R1595-R1604, 2000;
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Vol. 278, Issue 6, R1595-R1604, June 2000

Subthreshold aortic nerve inputs to neurons in nucleus of the solitary tract

Jing Zhang and Steven W. Mifflin

Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7764


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subthreshold aortic nerve (AN) inputs to neurons receiving a monosynaptic AN-evoked input (MSNs: respond to each of two AN stimuli separated by 5 ms) and neurons receiving a polysynaptic AN input (PSNs) in the nucleus of the solitary tract (NTS) were identified in anesthetized rats. In extracellular recordings from 24 MSNs and 49 PSNs, 12% of MSNs and 29% of PSNs only responded to AN stimulation during the application of excitatory amino acids. In intracellular recordings from 24 MSNs and 22 PSNs, 12% of MSNs and 14% of PSNs responded to AN stimulation with excitatory postsynaptic potentials that did not evoke action potential discharge. Reductions in arterial pressure produced minimal changes in the spontaneous discharge of suprathreshold AN-evoked neurons, suggesting that these neurons receive excitatory inputs from nonbaroreceptor sources. The results suggest that some baroreflex-related NTS neurons exist in a "reserve state" and can be changed to an active state or vice versa. This will change the number of neurons involved in baroreflex circuits and provides a novel mechanism for regulating baroreflex function independently of alterations in peripheral afferent input.

electrophysiology; baroreceptor; baroreflex


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OUR UNDERSTANDING OF BAROREFLEX function and attempts to model the baroreceptor reflex are critically dependent upon information regarding the number of neurons participating in the reflex at any given point in time. At resting levels of arterial pressure, the majority of arterial baroreceptors are above their threshold for action potential generation (4, 5, 28, 30, 35). Therefore, it is reasonable to assume that, when arterial pressure is at resting levels, the majority of baroreceptor afferents are providing a tonic, excitatory input to neurons in the nucleus of the solitary tract (NTS), where these peripheral afferents make their initial synapses. A simple model would assume that, at resting levels of arterial pressure, the majority of NTS neurons receiving an excitatory baroreceptor input are discharging action potentials as a result of this input. Increases or reductions in arterial pressure would then be encoded by either increases or decreases in the action potential discharge frequency of these neurons and/or the recruitment of additional neurons by higher threshold baroreceptor afferent inputs during increases in pressure.

Numerous studies have shown that electrical stimulation in a number of central sites can increase or decrease the action potential discharge frequency of NTS neurons (1, 3, 6, 7, 9, 10, 24, 33, 39). Therefore, these areas may alter baroreflex function by modulation of the discharge rate of NTS neurons that are already discharging action potentials under resting conditions. However, it is unknown whether modulatory inputs might also alter the number of neurons participating in the reflex at a given point in time, for example, by recruiting "silent" neurons that are not discharging action potentials under resting conditions. It is also unknown whether, under resting conditions, there are neurons that receive a peripheral baroreceptor afferent that does not result in action potential discharge.

Recently in our laboratory, NTS neurons receiving aortic nerve (AN) afferent inputs were observed during microiontophoretic application of excitatory amino acid (EAA) receptor agonists (37). We found that some NTS neurons that were not evoked by AN stimulation in the absence of EAAs became AN-evoked neurons when EAAs were applied. These neurons have been termed "subthreshold" neurons to differentiate them from suprathreshold neurons that respond to AN stimulation with an action potential. Based on these findings, we hypothesized that not all NTS neurons receiving baroreceptor afferent inputs necessarily participate in reflex function at resting levels of arterial pressure. There are any number of reasons that could underlie the existence of subthreshold neurons. Perhaps these neurons require a greater spatial or temporal facilitation of afferent input to reach threshold for action potential discharge. Perhaps the time-dependent and/or frequency-dependent inhibition described by Miles (17) and others (16, 27, 31) dampens excitatory transmission so that the input is not sufficient to evoke discharge. Perhaps some neurotransmitter (either excitatory or inhibitory) modulates neuronal excitability and shifts membrane potential to determine whether a neuron is subthreshold or suprathreshold.

A wide variety of neuromodulators, present within the NTS, could play a role in the modulation of baroreceptor afferent integration (13, 23, 32). EAAs have been suggested as the primary neurotransmitters mediating neurotransmission of the baroreflex within the NTS (8, 11, 12, 21, 29, 36-38). Several observations, however, suggest that EAAs may also act as modulators to influence neurotransmission by altering neuronal excitability. The first observation is that tonic EAA inputs contribute to the spontaneous discharge of both AN-evoked neurons and non-AN-evoked neurons (38), and of neurons with other functions (18, 34, 36). The second observation is that some AN-evoked neurons may receive tonic EAA inputs that are not from the AN. Microiontophoretic application of the non-N-methyl-D-aspartate (NMDA) EAA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 1,2,3,4-tetrahydro- 6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) selectively inhibited the spontaneous activity of some AN-evoked neurons without affecting the AN-evoked responses (38). This suggests that the excitatory drive to these neurons may be from non-AN inputs.

The experimental goals of the present study were twofold: 1) to identify and characterize NTS neurons receiving subthreshold AN inputs, and 2) to determine whether there are tonic EAA inputs to AN-evoked neurons that do not arise from peripheral baroreceptor afferent inputs.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General. Successful experiments were performed on 72 male Sprague-Dawley rats (350-500 g) (Charles River Laboratories, Willington, MA; or Harlan Sprague Dawley, Indianapolis, IN). Rats were housed two per cage in a fully accredited (American Association for Accreditation of Laboratory Animal Care and USDA) laboratory animal room with free access to food and water. All experimental rats were given at least 1 wk to acclimate before use. All experimental protocols were approved by the Institutional Animal Care and Use Committee.

Surgical preparation. Rats were initially anesthetized with pentobarbital sodium (60 mg/kg ip) and were placed on a thermostatically controlled heating pad. Body temperature was maintained at 36-38°C throughout the experiment. After placement of a venous catheter (tail vein) and cannulation of the trachea, the animal was artificially ventilated with oxygenated room air, and subsequent anesthetic was given as an infusion of 10-20 mg · kg-1 · h-1 iv. Gallamine triethiodide (20 mg · kg-1 · 30 min-1 iv) was given for paralysis to reduce respiratory movements of the brain stem. A femoral artery was cannulated for arterial blood pressure monitoring using a Cobe CDX transducer (Cobe Laboratories, Lakewood, CO). Mean arterial pressure and heart rate were determined from the pulsatile signal using a Coulburn blood pressure processor. Depth of anesthesia was assessed by monitoring the stability of arterial pressure and heart rate in response to pinch of the hind paw and was adjusted by appropriate changes in the infusion rate. The ANs were isolated bilaterally and marked with small pieces of black suture. The rat was placed in a stereotaxic head frame, and an occipital craniotomy was performed to expose the dorsal surface of medulla in the region of the obex. The AN ipsilateral to the central recording site was mounted on bipolar stimulating electrodes. The AN was stimulated with single pulses (0.5- to 1-ms pulse duration, 500 µA, 1 stimulus every 1.5 s). This stimulus intensity is sufficient to activate the entire population of afferent fibers in the AN (20, 26).

Intracellular recordings. Intracellular recordings were made using single-barrel electrodes filled with 2-4% Neurobiotin in 0.05 M Tris buffer (the tips of the electrode were <1 µm). All recordings were performed from 1.2 mm caudal and 0.5 mm rostral to the calamus scriptorius, 0-0.8 mm lateral to the midline, and 0.2-1 mm below the surface. The electrode was lowered into the tissue in 1-µm steps by a step-driver controller (Nanostepper; ALA Associates, Westbury, NY). To ensure that a good penetration was made, the membrane potential had to exceed 45 mV after 1-min observation, and the membrane potential drift had to be less than 5 mV during the data collecting period. Membrane potential, measured as the direct current (DC) potential recorded inside of the cell minus that recorded after withdrawal of the electrode from the cell, was amplified by a DC amplifier (World Precision Instruments, New Haven, CT) and sent to a digital oscilloscope (Nicolet Instrument, Madison, WI), an audiomonitor (Grass Instrument, Quincy, MA), a videotape recorder, and an analog-to-digital converter (model CED1401; Cambridge Electronic Design, Cambridge, UK) interfaced with a PC. Sigavg data acquisition software (Cambridge Electronic Design) was used for data collection and analysis. When an NTS neuron was successfully impaled and an AN-evoked excitatory or inhibitory postsynaptic potential (EPSP or IPSP, respectively) was observed, the AN input was characterized as monosynaptic (MSN) or polysynaptic (PSN) by the ability of MSNs to respond with an EPSP or action potential to each of two AN stimuli separated by 5 ms (17, 27, 37, 38). Most recorded cells were labeled by intracellular injection of neurobiotin and were examined later to verify that the recording site was within the NTS.

Extracellular recordings and microiontophoresis. Extracellular action potential discharge was recorded and microiontophoretic application of drugs performed with a five-barrel electrode (ASI Instrument, Warren, MI) as previously described (37, 38). The recording barrel was filled with a solution of 0.5 M sodium acetate containing 2% Chicago sky blue (impedance 8-30 MOmega ). One barrel of each five-barrel electrode was filled with a solution of 3 M NaCl and was used for automatic current balancing (Neurophore, Medical Systems). The remaining three barrels were filled with different drug solutions. The five-barrel electrode was lowered into the tissue in 2.0-2.5 µm steps by a step-driver controller (Burleigh Instrument, Fishers, NY), and a constant ejection current of 3-20 nA, to iontophorese EAA, was applied while lowering the electrode until a neuron was identified that responded to AN stimulation. Subthreshold AN-evoked neurons were identified as neurons that were no longer evoked by AN stimulation after stopping the EAA application. Suprathreshold AN-evoked neurons were identified as neurons in which action potential discharge was evoked in the absence of EAA application. The signals from the DC amplifier were sent to an alternating current (AC) filter, and then to the digital oscilloscope, audiomonitor, videotape recorder, and window discriminator (World Precision Instruments). The window discriminator output was led to the Cambridge Electronic Design analog-to-digital converter interfaced with the PC. Spike2 data acquisition software (Cambridge Electronic Design) was used for online and offline analysis. Peristimulus time (PST) histograms (0.5-s duration, 1-ms bin width, 40 sweeps) and rate meter histograms (180-s duration, 1-s bin width) were collected to analyze AN-evoked and spontaneous discharge, respectively. MSNs were differentiated from PSNs by the ability of MSNs to respond with an action potential to each of two AN stimuli separated by 5 ms.

Drugs were administered by application of microiontophoretic ejecting currents to the drug-containing barrels. After finding an AN-evoked neuron, EAA application was turned off to determine whether the neuron received a subthreshold AN input. Three to six PST histograms were collected, including 1-2 control PST histograms, 1-2 PST histograms during the EAA application, and 1-2 PST histograms after EAA application. To examine drug-induced effects on spontaneous discharge, drugs were ejected for successive 10- to 20-s periods separated by 20- to 80-s intervals until a steady-state level of excitation or inhibition was achieved. The drug solutions for microiontophoresis were: L-glutamic acid (monosodium salt, 100 mM; Sigma Chemical, St. Louis, MO), NMDA (100 mM; RBI, Natick, MA), (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 10 mM; Tocris Neuramin, Bristol, UK), kainic acid (10 mM; Sigma), CNQX (5 mM; RBI), and GABA (2 mM, pH 4.5; RBI). All drugs were dissolved in 150 mM saline and pH adjusted to 8.0-8.5, except GABA, whose pH value was adjusted to 4.0-4.5. All drugs were ejected as anions, except GABA, which was ejected as a cation. Retaining currents of appropriate polarity were applied to the drug barrels to retard the passive diffusion of the drug from the electrode tip during nonejection periods.

Data analysis. Data were analyzed with MANOVA (ANOVA) or ANCOVA with a repeated-measure design (StatSoft, Tulsa, OK). Newman-Keuls test was used for post hoc comparisons. Chi-square (chi 2) was used to analyze differences in population groupings. PST histograms were used to analyze evoked discharge. Response onset latency was defined as the interval between the stimulus artifact and the bin containing the greatest number of action potentials. Response onset latency variability was measured as the time interval between the minimum and maximum onset latency of the evoked responses recorded in a single PST histogram. Ratemeter records (spikes/s) were used for statistical analysis of spontaneous discharge. To account for different levels of basal spontaneous discharge, the firing rate increase (equal to the firing rate during the drug application minus baseline firing rate) was used for analysis. All values are expressed as means ± SE, except where indicated otherwise, and significance was accepted at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subthreshold AN-evoked NTS neurons identified by application of EAAs. In extracellular recording experiments, data were obtained from 73 NTS neurons; 56 were suprathreshold AN-evoked neurons, and 17 were subthreshold AN-evoked neurons. It is important to note that both subthreshold and suprathreshold neurons were found in a given experiment, even in the same electrode track in a given animal. MSNs and PSNs in the subthreshold group were initially identified during the application of EAAs and subsequently by the absence of AN-evoked discharge in the absence of EAA application. The specific EAA agonists and the cells tested were glutamate (2 MSNs, 4 PSNs), AMPA (1 MSN, 8 PSNs), kainate (1 PSN), and NMDA (1 PSN).

Figure 1 illustrates examples of a subthreshold MSN (Fig. 1A) and PSN (Fig. 1B). Neither neuron was spontaneously active and neither responded to AN stimulation in the absence of iontophoretic application of EAA agonist (Fig. 1, A1 and B1). During the application of glutamate, both neurons exhibited spontaneous discharge and AN-evoked discharge (Fig. 1, A2 and B2). After cessation of the iontophoresis, AN stimulation again failed to evoke discharge (Fig. 1, A3 and B3).


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  		Fig. 1.   Identification of subthreshold aortic nerve (AN)-evoked neurons in the nucleus of the solitary tract (NTS). A: poststimulus time histograms (PSTHs) illustrate a subthreshold monosynaptic AN-evoked input (MSN) identified by microiontophoretic application of glutamate. *AN stimulus artifacts. This MSN was not evoked by AN stimulation (A1) in absence of glutamate. Microiontophoretic application of glutamate induced AN-evoked responses and also increased the spontaneous discharge of this MSN (A2, iontophoretic ejection current is given in nA). The AN-evoked responses disappeared after stopping glutamate application (A3). B: a subthreshold polysynaptic AN-evoked input identified by glutamate application. The AN-evoked responses of this neuron were only apparent during application of glutamate (B2).

Among the 56 suprathreshold neurons, 21 were identified as MSNs and 35 were identified as PSNs. Among the 17 subthreshold neurons, 3 were identified as MSNs (3/24, 12%), and 14 neurons were PSNs (14/49, 29%). The EAA ejection currents necessary to change a subthreshold neuron to an AN-evoked neuron ranged from 3 to 20 nA. Microiontophoretic application of EAAs resulted in AN-evoked discharge and produced an increase in the spontaneous discharge of 15 subthreshold neurons (2 MSNs and 13 PSNs; 3 of these PSNs exhibited spontaneous discharge in the absence of EAAs). In these 15 subthreshold neurons, the application of EAAs increased spontaneous discharge from 0.7 ± 0.4 Hz (range 0-5.1 Hz, n = 15 including both MSNs and PSNs) to 6.2 ± 1.0 Hz (range 0-13.3 Hz) (P < 0.01). The AN-evoked responses were observed during EAA application for all 17 subthreshold cells (from 1.0 ± 0.4 spikes/40 stimuli for control to 22.9 ± 2.3 spikes/40 stimuli during the EAA application, P < 0.01).

The conversion of a subthreshold to a suprathreshold neuron by iontophoretic application of EAA agonist was also observed in the absence of any alteration in spontaneous discharge. Figure 2 illustrates examples where spontaneous discharge was not influenced by glutamate, but AN-evoked responses were induced by the same current ejection of the drug. This was observed in two neurons, a MSN (Fig. 2A) and a PSN (Fig. 2B).


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  		Fig. 2.   Glutamate can change subthreshold AN-evoked NTS neurons to suprathreshold neurons without affecting spontaneous discharge. A: PSTHs illustrate a subthreshold MSN identified by microiontophoretic application of glutamate. *AN stimulus artifacts. This MSN was not evoked by AN stimulation (A1) in the absence of glutamate. Microiontophoretic application of glutamate induced AN-evoked responses and did not induce spontaneous discharge in this neuron (A2, iontophoretic ejection current is given in nA). The AN-evoked responses disappeared after stopping glutamate application (A3). B: a subthreshold PSN identified by glutamate application. The AN-evoked responses of this neuron were only apparent during application of glutamate (B2). This ejection current of glutamate did not alter spontaneous discharge.

Several differences were found between the AN-evoked responses of supra- and subthreshold neurons. First, fewer (chi 2, P < 0.01) subthreshold neurons were spontaneously discharging action potentials (3/17, 18%; the 3 spontaneously active cells were PSNs) than suprathreshold neurons (35/56, 62%). The second difference is that the onset latencies for AN-evoked responses were more variable in subthreshold neurons (Fig. 3A). The average onset latency variability of subthreshold MSNs (14.0 ± 3.5 ms, range 10-21 ms, n = 3) and subthreshold PSNs (17.8 ± 2.7 ms, range 5-40 ms, n = 14) was significantly greater (P < 0.01) than the variability observed in suprathreshold neurons (5.0 ± 0.8 for MSNs and 9.6 ± 1.2 ms for PSNs). Within the suprathreshold groups the response onset latency variability was greater in PSNs than in MSNs (Fig. 3A).


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  		Fig. 3.   Comparison of onset latency variability and peak latency between sub- and suprathreshold AN-evoked neurons. A: comparison of the onset latency variabilities between suprathreshold (open bars) and subthreshold (solid bars) neurons for monosynaptically and polysynaptically evoked neurons. B: comparison of the peak latencies between suprathreshold (open bars) and subthreshold (solid bars) neurons for mono- and polysynaptically evoked neurons. *P < 0.05 vs. suprathreshold neurons. #P < 0.05 vs. monosynaptic neurons.

The average onset latencies for MSNs (19 ± 2 ms, range 4-27 ms) were less than that of PSNs (26 ± 1 ms, range 8-45 ms) (P < 0.01); however, there was considerable overlap between the two groups. The average onset latencies for subthreshold MSNs (18 ± 7 ms) and for subthreshold PSNs (26 ± 3 ms) were not significantly different (P = 0.79 and P = 0.66, respectively) from those of suprathreshold neurons (19 ± 2 ms for MSNs, 25 ± 1 ms for PSNs) (Fig. 3B).

Modulation of excitability by inhibitory agents was examined in 9 suprathreshold MSNs and in 11 suprathreshold PSNs (Fig. 4, these neurons are not included in the totals provided in the first paragraph of this section). Microiontophoretic application of GABA (2 mM) inhibited both the spontaneous discharge and the AN-evoked responses of these neurons (Fig. 4, A and B). During the application of GABA, these NTS neurons appeared similar to the subthreshold neurons. During iontophoresis of GABA, the neurons could be changed to suprathreshold neurons again by co-iontophoresis of glutamate (n = 1) or AMPA (n = 1) (Fig. 4C).


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  		Fig. 4.   An example of a suprathreshold AN-evoked neuron that was changed to a subthreshold neuron by application of GABA. This neuron was a PSN (A). Both spontaneous and AN-evoked discharge were inhibited by GABA (B), and this neuron became suprathreshold neuron again by coapplication of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (C). The GABA-evoked inhibition recovered after cessation of the AMPA iontophoresis (D), and both spontaneous and evoked responses recovered after cessation of the GABA iontophoresis (E).

Subthreshold AN-evoked neurons identified by intracellular studies. Data were obtained from 46 AN-evoked NTS neurons (24 MSNs and 22 PSNs). Three AN-evoked MSNs (3/24, 12%) and three AN-evoked PSNs (3/22, 14%) responded to AN stimulation with only an EPSP or EPSP-IPSP (only one MSN responded with an EPSP-IPSP) and no action potential. The remaining neurons responded with an EPSP or EPSP-IPSP and 1-9 action potentials arising from the EPSP. Subthreshold neurons could become suprathreshold neurons when depolarized using intracellular current injection (Fig. 5A). Conversely, suprathreshold neurons could become subthreshold neurons when hyperpolarized using intracellular current injection (Fig. 5B).


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  		Fig. 5.   Subthreshold AN-evoked NTS neurons illustrated by intracellular recordings. A: a subthreshold PSN that responded to AN stimulation with only an excitatory postsynaptic potential EPSP (top trace). This neuron was changed to a suprathreshold AN-evoked neuron by delivering the AN stimulus during a depolarizing current pulse (bottom trace). *AN stimulus artifacts. B: a suprathreshold PSN responding to AN stimulation with excitatory postsynaptic potential (EPSP; 2 EPSPs) and action potentials (5 action potentials) (top trace). This neuron was changed to a subthreshold neuron by delivering the AN stimulus during a hyperpolarizing current pulse (bottom trace).

Figure 6 illustrates comparisons of several parameters measured during intracellular recordings in subthreshold and suprathreshold neurons. The resting membrane potential of subthreshold MSNs (67.9 ± 5.7 mV) was significantly greater than that of suprathreshold MSNs (53.0 ± 1.7 mV, P = 0.006) (Fig. 6A). Although not statistically significant, a similar trend was observed in PSNs (membrane potentials of 64.9 ± 6.8 mV for subthreshold PSNs vs. 53.7 ± 2.2 mV for suprathreshold PSNs, P = 0.086). Unfortunately, the high-resistance electrodes used to impale small NTS neurons in vivo do not consistently pass current very well; therefore, it was not possible to reliably quantify neuronal input resistance. The amplitudes of AN-evoked EPSPs in subthreshold neurons (7.9 ± 3.6 for MSNs and 7.4 ± 2.7 mV for PSNs) were not different compared with those in suprathreshold neurons (6.5 ± 0.5 for MSNs and 7.6 ± 0.9 mV for PSNs, P = 0.29 and 0.92, respectively) (Fig. 6B). The durations of AN-evoked EPSPs in subthreshold neurons (10.0 ± 2.2 for MSNs and 25.5 ± 2.5 ms for PSNs) were also not different compared with those in suprathreshold neurons (8.9 ± 1.1 for MSNs and 22.4 ± 1.7 ms for PSNs). However, EPSP duration was significantly greater in PSNs compared with MSNs, in both the subthreshold and suprathreshold groups (Fig. 6C).


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  		Fig. 6.   Comparison of different parameters between sub- and suprathreshold neurons. A: comparison of resting membrane potentials between suprathreshold (open bars) and subthreshold (solid bars) neurons (*P < 0.05 vs. suprathreshold neurons) for monosynaptically and polysynaptically evoked neurons. B: comparison of EPSP amplitude between suprathreshold (open bars) and subthreshold (solid bars) neurons for mono- and polysynaptically evoked neurons. C: comparison of EPSP duration between suprathreshold (open bars) and subthreshold (solid bars) neurons for mono- and polysynaptically evoked neurons (#P < 0.05 vs. monosynaptic neurons).

Responses of MSNs to changes in arterial pressure. The question of whether there are some EAA inputs to AN-evoked neurons that do not originate from arterial baroreceptors was tested on four MSNs with spontaneous activity. PSNs were not tested because of their inconsistent responses to blood pressure changes (unpublished data). All four MSNs responded to phenylephrine (2-4 µg/kg iv)-induced blood pressure increases of 20-50 mmHg with increases in discharge frequency of 10-40 Hz (Fig. 7A). Unloading of the arterial baroreceptors by decreasing blood pressure 35-60 mmHg by intravenous nitroprusside (20-50 µg/kg) induced moderate reductions in spontaneous discharge frequency of 1-3 Hz in two of four MSNs (Fig. 7B). The spontaneous discharge of these two MSNs was inhibited by iontophoretic application of CNQX. CNQX-induced inhibition of spontaneous discharge (88% and 65%) was greater than that induced by baroreceptor unloading (16% and 19%) (Fig. 7C). Microiontophoretic application of CNQX inhibited the spontaneous discharge of one of the two MSNs whose spontaneous discharge was not altered during baroreceptor unloading.


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  		Fig. 7.   Comparison of excitatory amino acid (EAA) antagonist-induced inhibition on an MSN and the inhibition induced by unloading of the baroreceptors on the same neuron. Ratemeter record of discharge rate (DR) in 1-ms bins is at top and blood pressure (BP) is at bottom. This neuron responded to a blood pressure increase induced by intravenous phenylephrine (4 µg/kg) (A). Unloading of the arterial baroreceptors by intravenous nitroprusside (40 µg/kg) mildly decreased the firing rate of this neuron (B). Microiontophoretic application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (5 mM, 40 nA) virtually abolished the spontaneous discharge of this neuron (C).

Location of recorded cells. Figure 8 illustrates the location within the NTS of the cells recorded in the extracellular and intracellular studies. All recordings were at levels between 0.4 mm rostral to calamus and 1.2 mm caudal to calamus. There was no differential subnuclear localization of any specific group (MSN vs. PSN; subthreshold vs. suprathreshold) within the NTS.


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  		Fig. 8.   Reconstruction of recording sites obtained from extracellular recordings (left side of brain) and intracellular recordings (right side of brain). Group mean points illustrate the mean mediolateral and dorsoventral locations; bars represent the standard deviations of these values. In groups where the number of neurons was too small (extracellular: MSN subthreshold group; intracellular: MSN and PSN subthreshold groups), the individual recording sites are plotted. NTS, nucleus of the solitary tract; ts, solitary tract; DMNX, dorsal motor nucleus of the vagus; and XII, hypoglossal nucleus. Calibration bar in bottom right equals 200 µm. [Adapted from Paxinos and Watson (22).]


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have shown that the discharge frequency of NTS neurons can be modulated by activation of inputs to NTS from other central sites (1, 3, 6, 7, 9, 10, 24, 33, 39). However, these analyses have focused on the modulation of neurons that are spontaneously discharging action potentials under resting conditions and in response to activation of peripheral afferent inputs. The possibility that some NTS neurons receive a subthreshold afferent input and that pharmacological modulation of neuronal excitability can reveal the subthreshold input has never been demonstrated.

The present study demonstrates that a certain percentage of NTS neurons receive subthreshold AN inputs under resting conditions. These subthreshold neurons can be modulated by EAAs and converted to suprathreshold neurons; conversely, suprathreshold neurons can be modulated by inhibitory amino acids and converted to subthreshold neurons. The present study also suggests that MSNs receiving synaptic EAA inputs from the AN may also receive synaptic EAA inputs from nonbaroreceptor sources, possibly nonbaroreceptor peripheral afferents and/or inputs from other central sites. Such non-AN EAA inputs to MSNs could play an important role in the integration of AN afferents as they will influence neuronal responsiveness to a baroreceptor input. Taken together, both findings indicate an effective means of altering the number of neurons participating in regulatory circuits, and thereby reflex function, independently of any alteration in the level of the peripheral baroreceptor afferent input.

Subthreshold AN inputs to NTS neurons. The numbers of suprathreshold (AN-evoked neurons responding with action potentials) and subthreshold (AN-evoked neurons responding with only EPSPs) neurons reported in the present study are not likely to represent the relative proportion of these two groups of NTS neurons for the following reasons. It is very likely that some subthreshold neurons were not found during our extracellular recordings. The iontophoretic dose of EAA or the duration of application of EAA may have been insufficient to reveal some subthreshold neurons.

In addition, a small percentage of NTS neurons are not excited by iontophoretically applied EAAs (37). As a result, some subthreshold neurons may not be found, because of their insensitivity to EAAs. Along this line, in the extracellular studies, fewer MSNs than PSNs were found in the subthreshold group. One possible reason for this observation could be the lower sensitivity to EAA agonists of some MSNs compared with PSNs (37). In contrast to the extracellular study, the percentage of subthreshold neurons was found to be comparable in the MSN (12%) and PSN (14%) groups in the intracellular study. It is also important to remember that the ratio of subthreshold to suprathreshold neurons could be altered by the presence of anesthesia.

The mechanism(s) responsible for the existence of subthreshold AN-evoked neurons is currently not known. The subthreshold neurons receive similar AN inputs as suprathreshold neurons, as demonstrated by the fact that both groups have similar latencies for AN-evoked responses and similar amplitude and duration AN-evoked EPSPs. The similar amplitude and duration EPSPs suggests that presynaptic inhibition of AN-evoked transmitter release does not play a significant role in determining whether a neuron is sub- or suprathreshold. Subthreshold neurons are relatively hyperpolarized and this could be due to tonic, postsynaptic inhibitory inputs. Previous iontophoretic studies have demonstrated tonic GABA-ergic inhibition of most NTS neurons (2, 9). Alternatively, subthreshold neurons may receive fewer excitatory inputs under resting conditions. Several groups have used double pulse stimulation of afferent nerves, separated by 2 ms, to study afferent inputs to NTS neurons (2, 9), and it is possible that such temporal summation might be necessary to evoke action potential discharge in some NTS neurons.

In the present study, GABA and glutamate were used to manipulate neuronal excitability and examine AN-evoked inputs. This does not imply that these are the sole agents which could determine the balance between subthreshold and suprathreshold behavior. Any, or all, of the transmitters/modulators localized within the NTS (e.g., catecholamines, peptides) could play a role in the modulation of neuronal excitability and thereby responsiveness to afferent inputs (13, 23, 32). A goal for future work is to determine the peripheral and/or central site of origin of the various modulatory inputs and under what conditions do specific modulators play a physiologically relevant role.

The functional role of subthreshold AN-evoked neurons could be that they comprise a reserve pool of cells capable of participating in reflex regulatory circuits under certain conditions. Neurons that receive subthreshold AN inputs and have no spontaneous discharge are not likely to transfer information to higher order neurons under our experimental conditions. When the excitability of these neurons is changed by receiving more excitatory inputs or less inhibitory inputs, they can become suprathreshold neurons and participate in reflex regulatory circuits. If a baroreceptor afferent input has to traverse several synapses within the NTS before being relayed to other central sites involved in cardiovascular regulation, then a small percentage change in the total number of cells participating in the reflex at a given level of transmission, e.g., the MSN population, could have a profound effect on the number of cells participating at later stages and/or the discharge of cells already participating. This mechanism for reflex modulation can occur independently of any alteration in the level of the peripheral afferent input to the cells.

EAA inputs to AN-evoked NTS neurons. EAAs are important neurotransmitters in baroreceptor afferent integration in the NTS as demonstrated by in vivo studies (8, 11, 12, 21, 29, 36-38). Consideration of the role of EAAs in afferent integration within NTS has focussed on findings that indicate that an EAA is the neurotransmitter by which baroreceptor afferents activate NTS neurons and by which these second-order neurons relay the baroreceptor input to higher order neurons. However, another mechanism whereby EAAs might play a role in afferent integration within NTS is that an EAA may be the neurotransmitter utilized by non-AN or nonbaroreceptor afferent inputs to NTS neurons and could thereby act as a modulator of the AN-evoked input by changing neuronal excitability.

The present study found that reductions in arterial pressure to 50 mmHg, which should silence most arterial baroreceptors (4, 5, 28, 30, 35), produced very weak (0-19%) reductions in the spontaneous discharge frequency of MSNs. This raises the possibility that spontaneous discharge in some MSNs is primarily determined by inputs that are insensitive to the level of arterial pressure. Furthermore, these inputs are mediated by EAAs, as non-NMDA antagonists dramatically reduce spontaneous discharge (38 and present study). Baroreceptor unloading only mildly inhibited two of four AN-evoked neurons, indicating that some AN-evoked neurons do receive a tonic, albeit weak, baroreceptor input. The spontaneous discharge of the other MSNs was insensitive to changes in arterial pressure. Baroreceptor unloading may have been associated with reductions in carotid blood flow and thereby increases in chemoreceptor discharge. If a neuron received convergent baroreceptor and chemoreceptor excitatory inputs (15, 16), then the loss of baroreceptor excitation may have been offset by an increase in chemoreceptor excitation so that no change in overall discharge was observed. We cannot definitively eliminate this possibility; however, it is considered unlikely as such convergence was observed in a small percentage of NTS neurons and the temporal sequence of decreased baroreceptor afferent discharge and increased chemoreceptor afferent discharge would have to perfectly coincide.

The sources of the excitatory inputs that mediate the spontaneous discharge of AN-evoked neurons are currently not known. Since spontaneous discharge can be markedly reduced by EAA antagonists (38 and present study), it would appear that a large component of spontaneous discharge arises from excitatory synaptic inputs. Peripheral afferent inputs that could mediate this tonic EAA input arise from a variety of sites. The arterial chemoreceptors (15, 16), somatic afferents (31), and vagus (25) and laryngeal nerve afferents (6, 16) converge on some AN-evoked or baroreflex-related NTS neurons. Munch (19) described a group of "autoactive" aortic baroreceptors in the rat that discharged action potentials even when pressure was below threshold; therefore, even at very low pressures, some NTS neurons could receive a tonic baroreceptor input. Central sites that could contribute to the tonic EAA input are equally numerous (14). It is also important to consider that a number of state-dependent variables (e.g., level of anesthesia, blood gas levels, level of blood pressure) will influence the degree of activation of convergent peripheral and central inputs to NTS neurons receiving AN inputs. Some of these afferents use EAAs as the neurotransmitters for synaptic transmission to NTS neurons (18, 34, 36). Convergent peripheral and/or central afferent inputs, acting via EAAs or any other neuromodulator, may serve as modulators of the baroreceptor afferent input to NTS neurons. By modulating membrane potential, these convergent inputs could play a role in determining the number of subthreshold vs. suprathreshold neurons.

Perspectives

Some second-order and higher order NTS neurons receive subthreshold AN inputs and exist in a "reserve state" under certain conditions. The reserve or active status of these neurons is determined by the excitability of the neurons as reflected in their membrane potentials. Subthreshold AN-evoked NTS neurons can be modulated by EAAs and changed to suprathreshold AN-evoked NTS neurons. This change from "reserve" to active status will change the number of neurons involved in baroreflex circuits. This provides a novel mechanism for regulating reflex function independently of any alteration in the level of peripheral afferent input. It appears that the spontaneous discharge of AN-evoked neurons is not solely determined by peripheral baroreceptor afferent inputs. These nonbaroreceptor EAA inputs could play an important role in baroreflex regulation by modulating NTS neuronal responsiveness to baroreceptor afferent inputs. Finally, descending modulation of baroreflex gain might be accomplished by not only modulating the discharge of NTS neurons but also by altering the number of neurons participating in the reflex.


    ACKNOWLEDGEMENTS

We thank M. Vitela and M. Herrera-Rosales for expert technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-56637.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Zhang, Dept. of Pharmacology, Univ. of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7764 (E-mail: ZHANGJ{at}UTHSCSA.edu).

Received 24 September 1999; accepted in final form 10 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 278(6):R1595-R1604
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