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Am J Physiol Heart Circ Physiol 277: H1350-H1360, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 4, H1350-H1360, October 1999

A presynaptic mechanism contributes to depression of autonomic signal transmission in NTS

Chao-Yin Chen1, John M. Horowitz2, and Ann C. Bonham1

1 Division of Cardiovascular Medicine and Department of Pharmacology and 2 Department of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

With increasing frequencies of autonomic afferent input to the nucleus tractus solitarii (NTS), postsynaptic responses are depressed. To test the hypothesis that a presynaptic mechanism contributes to this frequency-dependent depression, we used whole cell, voltage-clamp recordings in an NTS slice. First, we determined whether solitary tract stimulation (0.4-24 Hz) resulted in frequency-dependent depression of excitatory postsynaptic currents (EPSCs) in second-order neurons. Second, because decreases in presynaptic glutamate release result in a parallel depression of alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) receptor-mediated components of EPSCs, we determined whether the magnitude, time course, and recovery from the depression were the same in both EPSC components. Third, to determine whether AMPA receptor desensitization contributed, we examined the depression during cyclothiazide. EPSCs decreased in a frequency-dependent manner by up to 76% in second- and 92% in higher-order neurons. AMPA and NMDA EPSC components were depressed with the same magnitude (by 83% and 83%) and time constant (113 and 103 ms). The time constant for the recovery was also not different (1.2 and 0.8 s). Cyclothiazide did not affect synaptic depression at >= 3 Hz. The data suggest that presynaptic mechanism(s) at the first NTS synapse mediate frequency-dependent synaptic depression.

voltage-clamp; excitatory postsynaptic currents; alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid; N-methyl-D-aspartic acid; short-term plasticity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE NUCLEUS TRACTUS SOLITARII (NTS) is the first central nervous system site where the control of autonomic reflex function is coordinated (17, 30). Afferent fibers from autonomic visceral sensory receptors, including the arterial baroreceptors, make their first central synapse in the NTS, releasing glutamate as the primary excitatory neurotransmitter (1, 32). The NTS is organized somewhat topographically, with baroreceptor afferent fibers terminating largely in the intermediate and caudal NTS medial to the solitary tract (17, 18). One intriguing aspect of baroreceptor and general sensory afferent signal processing in this NTS region is that as the frequency of afferent input increases, the responses of the postsynaptic neurons (which might be expected to be proportional to the input frequency were the neurons simply following the afferent signals) begin to fall off. The fall off has been characterized as a relative reduction in the amplitude of evoked excitatory postsynaptic potentials (EPSPs) (2, 6, 7, 20, 40), as well as a relative decrease in the number of evoked action potentials (14, 16, 19, 26, 28). Frequency-dependent synaptic depression has been observed in the NTS both in the whole animal with intact autonomic afferent fibers (16, 19, 26, 28) and in brain stem slices containing the NTS and autonomic afferent fibers conveyed in the solitary tract (2, 6, 7, 14, 20, 40). The high frequency limits on the autonomic signal transmission likely serve to optimize information transfer within the central networks. Understanding the mechanism(s) could clarify how autonomic neural networks operate and may provide new information on the physiological relevance of frequency limits on the encoding of sensory information within central autonomic networks.

The contribution of presynaptic versus postsynaptic mechanisms underlying the frequency-dependent synaptic depression in the NTS has not been fully resolved. Schild and colleagues (27) developed a mathematical model predicting that at frequencies of afferent input to the NTS in the range of 10-20 Hz, presynaptic mechanisms contribute to synaptic depression, whereas at higher input frequencies, desensitization of postsynaptic alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor influences becomes more prominent. Miles (20) also proposed a presynaptic mechanism, suggesting that the frequency-dependent decreases in amplitudes of minimally evoked EPSPs in the NTS resulted from an increased number of response failures, consistent with a presynaptic locus. We proposed that, at least in the intact animal, glutamate activation of presynaptic metabotropic glutamate receptors contributes to the frequency limits on aortic baroreceptor signal transmission in the NTS (16). With recordings of changes in EPSP amplitudes or action potential firing, voltage-dependent changes in postsynaptic ion channels or dendritic filtering can contribute to frequency-dependent changes in the responses. In addition, frequency-dependent potentiation of local gamma -aminobutyric acid (GABA)-mediated inhibitory circuits may also contribute (9, 11). These circumstances make it difficult to resolve with certainty the contribution of presynaptic versus postsynaptic mechanisms.

In other networks, investigators have examined presynaptic mechanisms by taking advantage of the observation that decreases in presynaptic glutamate release result in a proportional decrease in the amplitude of AMPA and N-methyl-D-aspartic acid (NMDA) receptor-mediated components of synaptically evoked excitatory postsynaptic currents (EPSCs) (3, 12, 21, 36). For example, in hippocampal slices, Perkel and Nicoll (21) showed that pharmacologically mediated decreases in presynaptic glutamate release by the GABAB agonist baclofen resulted in a parallel reduction in the amplitude of the AMPA and NMDA components of EPSCs. More recently, von Gersdorff et al. (36) used the same strategy to demonstrate that frequency-dependent synaptic depression in the calyx of Held was presynaptically mediated.

We previously demonstrated that dual AMPA and NMDA receptor-mediated components also coexist in solitary tract-evoked EPSCs in second-order NTS neurons (4) as has also been shown in the NTS in a brain stem-cranial nerve explant preparation for vagal-evoked EPSCs (29). The components exhibited the same temporal separation, characteristic current-voltage (I-V) relationships, and sensitivity to selective non-NMDA and NMDA receptor antagonists as documented in other neural networks (12). In the present study, we took advantage of the dual AMPA and NMDA components of NTS EPSCs to determine the extent to which presynaptic mechanisms contribute to frequency-dependent synaptic depression between primary sensory afferent fibers and second-order NTS neurons. We first determined whether frequency-dependent depression could be shown under voltage-clamp conditions (to rule out the confounding contribution of postsynaptic voltage-dependent ion channels) and whether the frequency-dependent depression could be demonstrated specifically at second-order neurons (to rule out potential contributions of local circuits). Second, because decreases in presynaptic glutamate release lead to parallel reductions in AMPA and NMDA currents (3, 12, 21, 36), we determined whether the two components of solitary tract-evoked EPSCs were depressed proportionally, that is, to the same extent and with the same time course. Third, to independently examine the role of AMPA receptor desensitization, we compared the depression of solitary tract-evoked EPSCs in the presence and absence of cyclothiazide, a compound that prevents AMPA receptor desensitization (35). All experiments were performed with the GABAA antagonist bicuculline in the perfusate to rule out the potential contribution of inhibitory synapses.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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

Slice Preparation

Male Sprague-Dawley rats 3-4 wk old (60-120 g) were anesthetized with a combination of ketamine (35 mg/kg) and xylazine (2 mg/kg). After each rat was decapitated, the brain was rapidly exposed and submerged in ice-cold (<4°C) high-sucrose artificial cerebrospinal fluid (aCSF) that contained (mM) 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 220 sucrose, and 2 CaCl2, pH 7.4, when continuously bubbled with 95% O2-5% CO2 (300 mosmol/l). Brain stem coronal slices (250 µm thick) were cut with the Vibratome 1000 (Technical Products International, St. Louis, MO). Two slices that included the NTS, solitary tract, and area postrema were typically obtained from each rat. After incubation for 45 min at 37°C in high-sucrose aCSF, the slices were placed in room temperature normal aCSF that contained (mM) 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, and 2 CaCl2, pH 7.4, when continuously bubbled with 95% O2-5% CO2 (300 mosmol/l). During the experiments a single slice was transferred to the recording chamber, held in place with a nylon mesh, and continuously perfused with oxygenated aCSF at a rate of ~3 ml/min. All experiments were performed at room temperature.

Whole Cell, Voltage-Clamp Recording

Borosilicate glass electrodes were filled with aCSF solution containing (mM) 145 CsF, 5 NaCl, 1 MgCl2, 3 K-ATP, 0.2 Na-GTP, 10 EGTA, and 10 HEPES, pH 7.4 (300 mosmol/l). Whole cell recordings in NTS neurons were made with the Axoclamp 1D patch-clamp amplifier (Axon Instruments, Foster City, CA). Whole cell currents were filtered at 1-2 kHz, digitized at 2.5-10 kHz with the DigiData 1200 Interface (Axon Instruments), and stored in a 386 DX computer. The pipette resistance was 2.5-5 MOmega . Data were analyzed off-line using the pCLAMP6 software (Axon Instruments). Stimuli (1.6-10 V, 0.1-ms square wave pulses) were delivered through bipolar tungsten electrodes (25-µm tips separated by 80 µm) to the solitary tract ipsilateral to the recording site. To limit the voltage spread outside the confines of the solitary tract, we used low stimulating voltages of <= 10 V. Cells requiring larger voltages for activation were not further studied. We previously showed that solitary tract stimulation at voltages ranging from 1 to 25 V activated EPSCs (having both AMPA and NMDA receptor components) in second-order neurons in coronal slices and in horizontal slices in which the stimulating electrode was separated from the recording electrode by up to 1 mm (4).

All experiments were performed on neurons under voltage-clamp conditions with the membrane voltage-clamped at -60 mV. The seal resistance was always >1 GOmega , and the series resistance was no larger than 30 MOmega . Solitary tract-evoked EPSCs were pharmacologically isolated by constant perfusion with the specific GABAA receptor antagonist bicuculline (10 µM). Recognizing the difficulties in establishing criteria for determining whether a cell is activated over monosynaptic versus polysynaptic pathways, we classified a neuron as second order if an EPSC was evoked by each of the two solitary tract stimuli separated by 5 ms and had an onset variability of <= 0.5 ms (20). For each neuron, the onset latency was determined by averaging the onset latencies of EPSCs evoked by five consecutive solitary tract stimuli delivered at 0.2 Hz. The onset latencies of the EPSCs were measured from the stimulus artifact to the beginning of the EPSCs. The onset variability was defined as the difference between the shortest and the longest onset latency of the five EPSCs.

Protocols

Frequency-dependent depression of synaptic transmission. To test for frequency-dependent synaptic depression, trains of 32 solitary tract stimuli were delivered at frequencies of 0.4, 1, 3, 9, or 24 Hz in randomized order. The number of stimuli was kept constant to assure that changes in synaptic transmission depended on frequency rather than the number of stimuli delivered. In pilot studies we found that the extent of frequency depression is the same when the number of stimuli is kept constant as when the stimulus time is kept constant (14). A control EPSC was established by averaging the peak EPSC amplitudes evoked by five consecutive solitary tract stimuli delivered at 0.2 Hz before each train of stimuli at every frequency. In pilot studies (n = 3), solitary tract stimulation at 0.2 Hz showed no depression; the steady-state value over the last 10 stimuli was 103 ± 1% (mean ± SD) of the control EPSC, evoked by 5 consecutive solitary tract stimuli delivered at 0.2 Hz before the train. To determine the magnitude of synaptic depression, peak EPSC amplitudes were averaged over the last 10 of the 32 stimuli at every frequency and expressed as a percentage of the control EPSC.

Time course for synaptic depression and recovery. Because the frequency-dependent synaptic depression was greatest during 24-Hz solitary tract stimulation, further data on the time course and recovery were obtained at that frequency. A control EPSC was established by averaging the peak EPSC amplitudes evoked by 5 solitary tract stimuli at 0.2 Hz. Then a train of 32 stimuli was delivered at 24 Hz. After each control, EPSC was established and a train of stimuli at 24 Hz was delivered, a test stimulus was applied with a delay of 0.5, 1, 2, 3, 4, 5, 10, or up to 50 s in 5-s increments in randomized order.

To determine the time course for the synaptic depression, the peak amplitude of the EPSC evoked by each stimulus in the train was expressed as a percentage of the control EPSC and plotted for each consecutive stimulus. These data points were fit by a single exponential function, and the time constant for the depression (tau dep) of the EPSC amplitude was determined. We also described the time course for the depression as the time required for the EPSC amplitude to decrease to 50% of the maximal depression.

To determine the time course for the recovery, the peak amplitude of the EPSC evoked by each test stimulus (applied after every train) was expressed as a percentage of the control EPSC and plotted for each test stimulus. These data points were also fit by a single exponential function, and the time constant for the recovery (tau rec) was determined. We also described the time course for the recovery as the time required for the EPSC amplitude to recover to 90% of the maximal recovery.

Synaptic depression and recovery of AMPA and NMDA components of EPSCs. We compared the magnitude and time course for the depression as well as the time course for the recovery of the AMPA and NMDA components of the EPSCs evoked by solitary tract stimuli delivered at 24 Hz. Because of the nonlinear relationship of the NMDA conductance to voltage due to the voltage-dependent Mg2+ block, to optimize the detection of the NMDA currents it is necessary to either depolarize the neuron to voltages positive to -45 mV or remove Mg2+ from the perfusate. In the present study, we performed the studies in nominally Mg2+-free perfusate to allow us to simultaneously monitor both the AMPA and NMDA receptor currents in neurons that were operating near their resting membrane potential (-60 mV) during frequency-dependent depression. Under these conditions of nominally Mg2+-free perfusate, the early rising phase, measured at the EPSC peak, is predominantly the AMPA receptor-mediated component, and the current measured at 20 ms after the peak is predominantly the later NMDA component (4, 8, 12). We have confirmed in a previous study that these temporally separated components are AMPA and NMDA receptor mediated, respectively, by their distinctive I-V relationships and by selective antagonist sensitivity (12). Thus in nominally Mg2+-free perfusate with the membrane potential clamped at -60 mV, both AMPA- and NMDA-receptor-mediated EPSC components are proportional to the presynaptic release of glutamate.

We used the same protocol as was used to examine the time course for synaptic depression and subsequent recovery of the peak EPSC amplitude, except that here we removed Mg2+ from the perfusate. Briefly, each trial consisted of five stimuli delivered at 0.2 Hz (to establish control AMPA and NMDA components of the EPSCs) followed by a train of 32 stimuli at 24 Hz and then a test stimulus. During the train of stimuli, the AMPA component of the evoked EPSC was expressed as a percentage of the AMPA component of the control EPSC, and the NMDA EPSC component was expressed as a percentage of the NMDA component of the control EPSC. The percentage for each EPSC component was plotted for each consecutive stimulus in the train. After each control and train of stimuli, a test stimulus was applied. The amplitude of the AMPA and NMDA components of the EPSC evoked by each test stimulus was expressed as a percentage of the control EPSC components and plotted for each consecutive test stimulus.

AMPA receptor desensitization and frequency-dependent synaptic depression. To determine the extent to which AMPA receptor desensitization may contribute to frequency-dependent synaptic depression, we compared the peak EPSC amplitudes evoked by trains of 32 solitary tract stimuli delivered at 0.4, 3, 9, or 24 Hz before and during perfusion with cyclothiazide (0.1 mM).

Data Analyses

Data are expressed as means ± SE unless otherwise indicated. Differences were considered significant at P < 0.05. To compare the magnitude of the frequency-dependent synaptic depression, the steady-state EPSC amplitude (averaged over the last 10 stimuli and expressed as a percentage of the control EPSC) was compared in second- versus higher-order neurons at each frequency by a two-way ANOVA, followed by Fisher's least significant difference test. The tau dep for synaptic depression at second- versus higher-order neurons was compared during the 24-Hz stimulation by using an unpaired t-test. The tau rec was also compared by using an unpaired t-test.

The magnitude of the depression of AMPA- and NMDA-receptor-mediated EPSCs was analyzed by comparing the new steady-state amplitudes of each EPSC component with a paired t-test. The tau dep and tau rec of the AMPA and NMDA components were also compared by using a paired t-test.

The effect of cyclothiazide on frequency-dependent depression was evaluated by comparing the average steady-state EPSCs evoked at 0.4, 3, 9, or 24 Hz using a two-way ANOVA with frequency as the between factor and presence or absence of cyclothiazide as the within factor.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results are based on whole cell recordings of EPSCs evoked by solitary tract stimulation in neurons classified as second or as higher order. EPSCs evoked in neurons that met the presumptive criteria for second order (n = 30) had shorter onset latencies and tighter onset variabilities compared with EPSCs evoked in higher-order neurons (n = 52). Mean onset latency was 2.57 ± 0.18 and 3.76 ± 0.18 ms (P = 0.00004, unpaired t-test), and mean onset variability was 0.41 ± 0.02 and 0.99 ± 0.07 ms for monosynaptic and polysynaptic EPSCs, respectively (P = 0.00001, unpaired t-test). The mean decay time constant for individual EPSCs could be fit with a single exponential and was not different in second- and higher-order neurons (5.6 ± 0.3 vs. 6.3 ± 0.3 ms, respectively; P = 0.15, unpaired t-test). Stimulating voltages activating second- and higher-order neurons were not different (5.5 ± 0.5 vs. 5.2 ± 0.4 V, respectively; P = 0.57; unpaired t-test). Figure 1A shows the patch pipette attached to an NTS neuron. All neurons studied were located in the intermediate NTS at the level of the area postrema in the rostrocaudal plane and medial to the solitary tract, the region of the nucleus that receives a major input from baroreceptors and is essential for baroreflex function located in the medial aspects of the intermediate and caudal NTS (Fig. 1B) (17).


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  		Fig. 1.   Location of neurons in nucleus tractus solitarii (NTS) that displayed frequency-dependent synaptic depression of solitary tract-evoked excitatory postsynaptic potentials (EPSCs). A: photograph showing patch pipette attached to membrane surface of an NTS neuron for whole cell recordings. B: schematic drawing showing composite of recording sites of EPSCs evoked in second-order neurons (open circle ) and higher-order neurons (). All neurons were located in the intermediate NTS, medial to solitary tract (ST). AP, area postrema; C, central canal.

Frequency-Dependent Depression of Synaptic Transmission at Second- and Higher-Order NTS Neurons

The peak amplitudes of monosynaptically and polysynaptically evoked EPSCs were depressed in a frequency-dependent manner. Figure 2 shows an example of frequency-dependent depression of monosynaptically evoked EPSCs. The peak EPSC amplitude decreased progressively from the control EPSC during the train of stimuli applied at each frequency (Fig. 2A). As the trains of stimuli were delivered with increasing frequencies, this decrease in the peak EPSC amplitude became more prominent. In Fig. 2B for the same neuron, the decreases in the peak EPSC amplitudes are averaged for the last 10 stimuli at each stimulation frequency and expressed as a percentage of the control EPSC.


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  		Fig. 2.   Example of frequency-dependent depression of EPSCs evoked in a second-order neuron during 32 solitary tract stimuli at each frequency. A: in each set of traces, largest current is control EPSC (*) and each successive current is an average of 2 EPSCs evoked by successive stimuli. At each frequency the peak EPSC amplitude decreased progressively during the train of stimuli. As the trains were applied with increasing frequencies, peak EPSC amplitudes decreased to a greater extent. B: peak amplitude of EPSCs recorded from the neuron in A are averaged for the last 10 stimuli at each frequency and expressed as a percentage of control EPSC.

The group data (Fig. 3) illustrate that frequency-dependent depression of synaptic transmission occurred at both second- and higher-order neurons. The depression was less robust in second- than in higher-order neurons as indicated by the smaller decrease in the peak amplitudes of monosynaptically evoked EPSCs compared with polysynaptically evoked EPSCs during stimulation frequencies of 3-24 Hz.


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  		Fig. 3.   Group data showing frequency-dependent synaptic depression of EPSCs evoked in second-order neurons and higher-order neurons. Trains of 32 solitary tract stimuli delivered at 0.4, 1, 3, 9, or 24 Hz decreased peak amplitude of the EPSCs (averaged over the last 10 stimuli at each frequency and expressed as a percentage of control EPSC). Frequency-dependent depression was significantly greater in higher- than in second-order neurons beginning at 3 Hz. (two-way ANOVA; P < 0.0001, frequency; P < 0.0001, neuron order; P < 0.0001, frequency * neuron order).

Time Course for Synaptic Depression and Recovery

The time course for the synaptic depression during and the time course for the recovery after a train of solitary tract stimuli at 24 Hz are shown in Fig. 4. An example from one neuron is shown in Fig. 4, A and B. The amplitude of the peak EPSC evoked by successive stimuli decreased progressively (Fig. 4A, left). The peak EPSC amplitude was still depressed following a test stimulus at 0.5 and 1 s after a train of stimuli but recovered to control when a test stimulus was delivered at 5 and 10 s after a train of stimuli (Fig. 4A, right). The data points for the time course for the depression and for the recovery were best fit with a single exponential; tau dep was 56 ms, and the time required for 50% depression was 52 ms (Fig. 4B, left). tau rec was 0.6 s, and the time required for 90% recovery was 1.4 s (Fig. 4B, right). The time courses for the synaptic depression and subsequent recovery are summarized for the group data in Fig. 4C. In both second- (n = 8) and higher-order neurons (n = 16), peak EPSC amplitudes decreased with a time course best fit with a single exponential (Fig. 4C, left). The peak amplitude of EPSCs evoked in second-order neurons decreased to a new steady state (25.6 ± 6.6% of the control) with a tau dep of 94 ± 43 ms and a time to 50% depression of 65 ± 30 ms. In higher-order neurons, although the new steady-state peak EPSC amplitude (9.2 ± 2.9% of the control) was significantly lower than that in the second-order neurons (P = 0.01, unpaired t-test), neither the tau dep (91 ± 26 ms) nor the time to 50% depression (63 ± 18 ms) differed from the values obtained in the second-order neurons.


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  		Fig. 4.   Time course for synaptic depression during a 24-Hz train of 32 solitary tract stimuli and subsequent recovery. A, left: peak EPSC amplitude decreased progressively with each successive stimulus during the train. Right, at time intervals (Delta t) of 0.5, 1, 5, or 10 s after each train, a stimulus was delivered to test for recovery. Peak EPSC amplitude recovered when a stimulus was delivered at 5 or 10 s. B, left: peak amplitude of each EPSC evoked in the neuron in A is expressed as a percentage of control EPSC and plotted for each consecutive stimulus in the train. Right, for same neuron, recovery of peak EPSC amplitude evoked by each test stimulus is expressed as a percentage of control and plotted with respect to time after each train. C, left: group data showing peak amplitude of EPSCs evoked in second- (n = 8) and higher-order neurons (n = 16) decreased to new steady states with same time constant for depression (tau dep) (P = 0.95, unpaired t-test). Right, group data showing that the time constant for recovery (tau rec) was also not different in the two groups of neurons. (P = 0.8, unpaired t-test).

The recovery time course (Fig. 4C, right) was best fit with a single exponential and was not different in the two groups of neurons. The peak EPSC amplitude in second-order neurons recovered with a tau rec of 2.1 ± 0.7 s and to 90% of the control EPSC in 4.9 ± 1.7 s. In 15 of the higher-order neurons, the tau rec was 1.9 ± 0.6 s, and time for recovery to 90% of control was 4.1 ± 1.3 s. One EPSC evoked in a higher-order neuron failed to recover and was not included in the analysis.

Synaptic Depression and Recovery of AMPA- and NMDA-Receptor-Mediated EPSCs

The proportionate synaptic depression and recovery of AMPA- and NMDA-receptor-mediated EPSC components are shown in Fig. 5. Figure 5A shows the contour of the same EPSC evoked in normal perfusate and in a nominally Mg2+-free perfusate (where the block of the NMDA receptor channel is relieved); the early rising phase, measured at the EPSC peak is predominantly the AMPA component, and the current measured at 20 ms after the peak is predominantly the later NMDA component (4, 8, 12). As shown in Fig. 5B (left), the extent and time course for the synaptic depression of both the AMPA and NMDA EPSC components evoked in second-order neurons (n = 8) were the same. The AMPA component decreased to a steady-state value of 16.8 ± 3.9% of control, and the NMDA component decreased to 17.0 ± 3.3% (P = 0.9, paired t-test). The time course for the depression was best fit with a single exponential; tau dep was 113 ± 34 ms for the AMPA component and 103 ± 30 ms for the NMDA component. The time to 50% depression was 79 ± 23 ms for the AMPA component and 71 ± 21 ms for the NMDA component. The time courses for the recovery of the AMPA and NMDA components of the EPSCs were also not different in second-order neurons (Fig. 5B, right). The data were best fit with a single exponential; tau rec was 1.2 ± 0.4 s for the AMPA component and 0.81 ± 0.22 s for the NMDA component. The time for 90% recovery was 2.8 ± 0.8 and 1.9 ± 0.5 s for the AMPA and NMDA components, respectively.


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  		Fig. 5.   Synaptic depression and recovery of alpha -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartic acid (NMDA) EPSC components. A: average of 5 EPSCs evoked by 5 solitary tract stimuli delivered at 0.2 Hz in normal and in nominally Mg2+-free perfusate. B, left: time course for synaptic depression of AMPA and NMDA EPSC components in second-order neurons (n = 8). Amplitudes of both EPSC components decreased to same steady-state value (P = 0.92, paired t-test) and with the same tau dep (P = 0.90, paired t-test). Right: time course for recovery of AMPA and NMDA EPSC components. Both components recovered with same tau rec (P = 0.37, paired t-test). C, left: AMPA- and NMDA EPSC components evoked in higher-order neurons (n = 12) also decreased to same extent (P = 0.55, paired t-test) and with the same time course (P = 0.65, paired t-test). Recovery time course also was not different (right) (P = 0.96, paired t-test).

AMPA and NMDA EPSC components evoked in higher-order neurons also decreased to the same extent and at the same rate (Fig. 5C, left, n = 12). The AMPA component decreased to 7.7 ± 2.0% of control and the NMDA component to 8.7 ± 2.1% of control. The tau dep was 79 ± 18 and 89 ± 18 ms for the AMPA and NMDA components, respectively. The AMPA and NMDA components also recovered in parallel. The tau rec was 1.0 ± 0.2 s for the AMPA component and 1.0 ± 0.2 for the NMDA component (Fig. 5C, right).

As was the case when peak EPSC amplitudes were measured in normal perfusate (containing Mg2+), under conditions of nominally Mg2+-free perfusate the maximum decrease in the AMPA component of the EPSC was significantly greater in higher- than in second-order neurons (7.7 ± 2.0% of the control EPSC in higher-order and 16.8 ± 3.9% of control in second-order neurons; P = 0.04, unpaired t-test). The extent of the depression was the same when the peak EPSC was measured in normal perfusate and when the AMPA EPSC component was measured in nominally Mg2+-free perfusate (P = 0.23 for comparisons in second-order neurons and P = 0.69 for comparisons in higher-order neurons, unpaired t-test). The maximum decrease in the NMDA component was also significantly greater in higher- than in second-order neurons (8.7 ± 2.1% of control in higher- and 17.0 ± 3.3% of control in second-order neurons; P = 0.04, unpaired t-test).

AMPA Receptor Desensitization and Frequency-Dependent Synaptic Depression

The peak amplitude of EPSCs evoked by low-frequency solitary tract stimulation of 0.2 Hz was not affected by cyclothiazide, although as expected the decay time was prolonged (35) (Fig. 6A). The effect of cyclothiazide on frequency-dependent depression (n = 6) is shown in Fig. 6B. During solitary tract stimulation at 3, 9, or 24 Hz, where the synaptic depression became increasingly more pronounced, there was no significant change in the steady-state response when AMPA receptor desensitization was blocked. By contrast, during stimulation at 0.4 Hz, when synaptic depression was modest, there was a small, yet statistically significant reduction during cyclothiazide.


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  		Fig. 6.   Frequency-dependent synaptic depression before and during prevention of AMPA receptor desensitization. A: average of 5 EPSCs evoked by 5 solitary tract stimuli delivered at 0.2 Hz before and in presence of cyclothiazide. Peak EPSC amplitude was same, although decay was prolonged. B: in normal perfusate, peak EPSCs (averaged over last 10 stimuli at each frequency and expressed as a percentage of control) (open circle ) decreased in a frequency-dependent manner. Perfusion with cyclothiazide () had no effect on frequency-dependent synaptic depression at 3, 9, and 24 Hz but slightly reduced depression during 0. 4 Hz of stimulation. (2-way ANOVA; P < 0.0001, frequency; P = 0.0184, cyclothiazide; P = 0.68, frequency * cyclothiazide; P = 0.02, normal perfusate vs. cyclothiazide at 0. 4 Hz).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study provides new evidence consistent with the hypothesis that presynaptic mechanism(s) contribute to frequency-dependent depression of autonomic signal transmission in the NTS at input frequencies of 3-24 Hz and that the depression is initiated at the first central synapse between sensory afferent fibers and second-order neurons. The evidence is based on the findings that: 1) when the membrane potential of NTS neurons was voltage-clamped at -60 mV (eliminating influences of postsynaptic voltage-dependent mechanisms to frequency-dependent depression), the amplitudes of solitary tract-evoked EPSCs decreased in a frequency-dependent manner; 2) frequency-dependent depression of EPSCs was observed in neurons that met the criteria for monosynaptic activation (minimizing the potential contribution of postsynaptic changes in intervening neurons between the primary sensory afferent fibers and NTS neurons); and 3) both AMPA and NMDA components of EPSCs in second-order neurons were depressed and recovered in parallel [consistent with a presynaptically mediated decrease in glutamate release (3, 12, 21, 36)]. Additional data obtained with cyclothiazide provided additional, independent evidence ruling out one important postsynaptic mechanism, AMPA receptor desensitization, at least at frequencies between 3 and 24 Hz.

The most direct evidence for a presynaptic site at the first central synapses is the finding that the AMPA and NMDA components of the EPSCs in second-order neurons were depressed in parallel, both in magnitude and time course, and, furthermore, that the two components recovered with the same time course. If presynaptic glutamate release is altered, it follows that the amplitudes of the two components of the synaptically evoked EPSCs will change proportionally. This has been shown to be the case when presynaptic glutamate release is changed pharmacologically by theophylline or baclofen (21), by alterations in the Ca2+-to-Mg2+ ratio (3), or by frequency-dependent depression (36). Of particular relevance to the current study was the use of this strategy by Neher's group to demonstrate that frequency-dependent depression at the calyx of Held is presynaptically mediated (36). Comparing the magnitude and time course for the depression of the dual EPSC components is one of the most direct and feasible strategies for investigating presynaptic mechanisms in the NTS. An even more direct approach is voltage clamping both the presynaptic terminal fiber and postsynaptic neuron to simultaneously measure presynaptic and postsynaptic currents. Whereas this is possible in networks where the presynaptic terminal fibers are large such as the calyx of Held (31), the much smaller size of the sensory afferent fibers in the solitary tract limits the use of this technique in the NTS.

The coexistence of both AMPA and NMDA components in synaptically evoked EPSCs in second-order NTS neurons is a key to the strategy used in this study. We previously demonstrated the coexistence of these receptor components on second-order neurons based on their temporal separation, distinctive I-V relationships, and selective antagonist sensitivity (4, 12). Specifically, the early EPSC component (measured at the EPSC peak, see Fig. 5) displayed a linear relationship to voltage, characteristic of AMPA-receptor-mediated currents, whereas the slow EPSC component (measured at 20 ms after the peak) had a nonlinear relationship to voltage due to the voltage-dependent Mg2+ block at hyperpolarized membrane potentials, characteristic of NMDA-receptor-mediated currents. In addition, the selective non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) blocked only the early EPSC component while sparing the slow component, and the selective NMDA receptor antagonist APV selectively blocked the slow component while sparing the early component (4). Whole cell, voltage-clamp recordings in the NTS of a brain stem cranial nerve explant preparation has similarly shown that vagal transmission activates both non-NMDA and NMDA receptors on second-order NTS neurons (29). The dual receptors allow for the investigation of presynaptic glutamate release using voltage clamping. However, in terms of signal transmission, since activation of NMDA currents requires both glutamate and relief of the voltage-dependent Mg2+ block, when the postsynaptic neurons are near their resting membrane potential, activation of AMPA currents is sufficient to trigger action potentials and hence mediate synaptic transmission. This has been documented for low-frequency solitary tract stimulation in the horizontal slice by Andresen and Yang (1).

Central to the conclusion that a presynaptic mechanism contributes to frequency-dependent depression of autonomic signal transmission is that the depression occurs at the first NTS synapse between the sensory afferent fibers and second-order neurons. If the depression is only observed at higher-order neurons, postsynaptic changes at intervening synapses (in addition to changes at the presynaptic terminal at those more distal synapses) may contribute to the response of the neuron from which the recordings are made. Thus the presumptive nature of the criteria for distinguishing second- from higher-order neurons must be considered. Miles (20) was one of the first to emphasize the importance of establishing criteria beyond short onset latency for distinguishing the activation of second- from higher-order neurons in describing frequency-dependent depression in the NTS. Recognizing the difficulties in establishing criteria for monosynaptic activation, we used the two criteria used by Miles and presumed that a neuron was second-order if an EPSC was evoked by each of two solitary tract stimuli separated by 5 ms and was evoked with an onset variability of <= 0.5 ms. The first criterion has been validated by findings by Scheuer and Mifflin (25) that NTS neurons identified independently as second-order (by anterograde tracing of the vagus nerve) reliably met the criterion of following two stimuli separated by 5 ms. However, one important concern that may complicate the use of these criteria, particularly when measuring EPSPs or action potentials, is the potential effect of dendritic filtering. Under conditions when the membrane potential is not clamped, extensive dendritic spine density can result in a greater dendritic filtering (23), which might allow for a higher variability in the response latency and perhaps a failure to follow the twin stimuli, resulting in an erroneous classification of a second-order neuron as a higher-order neuron. However, under voltage-clamp conditions the effect of dendritic filtering is minimal as shown by Titz and Keller (34) in the NTS. In our study, the voltage-clamp conditions were judged adequate based on the criteria used by Hestrin et al. (12) that the I-V relationship of NMDA currents from whole cell recordings in the hippocampal slice were the same as the I-V relationship of currents through single NMDA channels in isolated cells. As pointed out by Hestrin, if the DC control was inadequate in the slice, the peak of the I-V relationship would be shifted to the left, a shift not seen in their study, in our previous study (4), or in pilot studies for this work, in which we used whole cell, voltage-clamp recordings to examine the I-V relationship of NMDA currents in NTS neurons. With the same voltage-clamp procedure, whole cell EPSCs recorded from NTS neurons in the current study displayed frequency-dependent synaptic depression (Figs. 2 and 3).

The experimental evidence obtained in this study for the contribution of a presynaptic mechanism in frequency-dependent depression occurring with input frequencies between 3 and 24 Hz is consistent with the predictions of the model by Schild and colleagues (27), with the proposal by Miles (20), and with our own data from in vivo studies (16). The relative contribution of the specific presynaptic mechanisms has yet to be resolved. Schild et al. (27) proposed effects of vesicle mobilization and depletion. Glaum and Miller (10) have shown in vitro that metabotropic glutamate receptors contribute to posttetanic EPSP depression. We also suggest that, at least in vivo, presynaptic metabotropic glutamate receptors play a role (37). Other possible presynaptic mechanisms include calcium current inhibition by mechanisms other than presynaptic metabotropic glutamate receptors (39) or adaptation of the calcium sensor for exocytosis (13).

Could postsynaptic mechanisms account for the parallel frequency-dependent depression and recovery of the AMPA and NMDA EPSC components in second-order neurons under voltage-clamp conditions? One possibility is that there is a proportional and synchronous desensitization of both AMPA and NMDA receptors. However, when AMPA receptor desensitization was prevented, the frequency-dependent depression persisted at input frequencies of 3-24 Hz, frequencies at which synaptic depression was most pronounced in this study, and at which synaptic depression has been observed in other studies in vitro (6, 7, 20) and in vivo (16, 19, 26, 28). Still, the small effect of cyclothiazide at 0.4 Hz (steady-state EPSC amplitude was 84% of the control EPSC before and rose to 92% during cyclothiazide) raises the possibility that AMPA receptor desensitization may play a small role in regulating synaptic throughput at low frequencies (<3 Hz) of sensory input. Other possible effects of cyclothiazide must be considered. Cyclothiazide may have slight presynaptic effects (35); however, they are minimal with extracellular Ca2+ concentrations of 2 mM used in the present study (38). Moreover, cyclothiazide did not alter the peak amplitude of the AMPA current (Fig. 6), further suggesting that any presynaptic effect was minimal. In addition, not all AMPA receptors may display cyclothiazide-sensitive desensitization (22), but this appears not to be the case in the NTS based on findings by Titz and Keller (34). NMDA receptor desensitization was not examined, however, if NMDA receptor desensitization was important, the NMDA receptor EPSC component would be expected to decay to a disproportionately greater extent than the AMPA receptor component. It did not.

Regarding frequency-dependent depression in higher-order neurons, because the higher-order neurons are voltage-clamped, the parallel depression and recovery of the AMPA and NMDA EPSC components indicate a presynaptic mechanism in that glutamate released from the presynaptic terminal (at that synapse) is decreased. However, given that we can voltage clamp only the last neuron in the path, the voltage at the intervening neurons can vary. Thus postsynaptic mechanisms in those intervening neurons, including opening (or closing) of voltage-dependent ion channels and/or dendritic filtering, could exert an effect before the signal arrives at the clamped higher-order neuron. Whether the frequency-dependent depression at the higher-order neurons is simply a reflection of presynaptic depression at multiple synapses or a combination of presynaptic and postsynaptic mechanisms cannot be resolved with certainty in this study.

Another possible postsynaptic mechanism that may be important at higher-order neurons is the potentiation of inhibitory responses during high-frequency solitary tract stimulation (10-50 Hz) that has been observed when NMDA and AMPA receptors were blocked (9, 11). These findings suggest that increases in inhibitory activity may also depress postsynaptic responses to sensory input. In the present study depression was observed with GABAA receptors blocked, but we cannot rule out the possibility that a buildup of some inhibitory factor, other than GABA, could have contributed.

Frequency-dependent synaptic depression occurs throughout the CNS (16, 21, 24, 36) and has been well documented in the NTS both in vitro (2, 6, 7, 14, 20, 40) and in the whole animal (16, 19, 26, 28). Miles (20), Champagnat et al. (6, 7), and Andresen and Yang (2) demonstrated frequency dependence of EPSPs when the solitary tract was stimulated at 0.5-100 Hz for 2-60 s. Zhou and co-workers (40), stimulating for 5 min, observed depression lasting several minutes. The frequency-dependent depression of EPSCs was similar to that observed previously for EPSPs. For example, in the Miles study (20), EPSP amplitudes decreased by 35% at 5 Hz, by 60% at 10 Hz, and by 80% at 20 Hz. Here, EPSC amplitudes in second-order neurons decreased by 37% at 3 Hz, by 57% at 9 Hz, and by 76% at 24 Hz.

In early in vivo studies, Seller and Illert (28) showed frequency-dependent synaptic depression of cardiorespiratory-related inputs conveyed by the carotid sinus nerve. More recently, with extracellular recordings, Scheuer et al. (26), using paired stimuli, and we, applying 100 (16), reported that baroreceptor signals conveyed by the aortic depressor nerve also show frequency-dependent depression in the NTS at input frequencies of 3-100 Hz, frequencies within the physiological range of baroreceptor activity; myelinated activity increases up to ~80 Hz for blood pressure increases of ~28 mmHg (5), and unmyelinated activity increases up to ~5 Hz with 40 mmHg pressure increases (33). In agreement with the current findings, frequency-dependent depression of aortic baroreceptor signal transmission is also more robust in higher- than in second-order neurons (16, 26). In that regard, the finding that there was a graded frequency-dependent depression in the EPSC amplitude rather than an all-or-none presence of an EPSC in the higher-order neurons suggests that within the NTS circuitry, one higher-order neuron does not receive input from only one second-order neuron, an arrangement consistent with the pattern of local connections proposed by Kawai and Senba (15) based on their morphological findings.

In summary, the results of this study suggest that the high-frequency limits on autonomic sensory signal transmission in the NTS are initiated, largely, by a presynaptic mechanism at the first synapse between the sensory afferent fiber and the second-order neuron. This presynaptic frequency-dependent depression, serving as a low-pass filter, allows incoming sensory, including baroreceptor, signals with a wide dynamic range (and hence less subject to noise) to be converted to signals having a smaller dynamic range (and hence more easily modulated by other inputs).


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance of Judy Stewart.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-52165 and HL-60560 and by the Health Systems Research Award from the University of California, Davis.

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: A. C. Bonham, Univ. of California, Davis, Division of Cardiovascular Medicine, TB 172 One Shields Ave., Davis, CA 95616 (E-mail: acbonham{at}ucdavis.edu).

Received 7 December 1998; accepted in final form 10 May 1999.


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METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 277(4):H1350-H1360
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