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J Appl Physiol 94: 819-827, 2003. First published October 25, 2002; doi:10.1152/japplphysiol.00552.2002
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Vol. 94, Issue 2, 819-827, February 2003

HIGHLIGHTED TOPICS
Plasticity in Respiratory Motor Control
Selected Contribution: Neuroplasticity in nucleus tractus solitarius neurons after episodic ozone exposure in infant primates

Chao-Yin Chen1, Ann C. Bonham1,2, Charles G. Plopper3, and Jesse P. Joad4

Departments of 1 Internal Medicine, 2 Pharmacology and Toxicology, and 4 Pediatrics, School of Medicine, and 3 Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California at Davis, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Acute ozone exposure evokes adverse respiratory responses, particularly in children. With repeated ozone exposures, however, despite the persistent lung inflammation and increased sensory nerve excitability, the central nervous system reflex responses, i.e., rapid shallow breathing and decreased lung function, adapt, suggesting changes in central nervous system signaling. We determined whether repeated ozone exposures altered the behavior of nucleus tractus solitarius (NTS) neurons where reflex respiratory motor outputs are first coordinated. Whole cell recordings were performed on NTS neurons in brain stem slices from infant monkeys exposed to filtered air or ozone (0.5 ppm, 8 h/day for 5 days every 14 days for 11 episodes). Although episodic ozone exposure depolarized the membrane potential, increased the membrane resistance, and increased neuronal spiking responses to depolarizing current injections (P < 0.05), it decreased the excitability to vagal sensory fiber activation (P < 0.05), suggesting a diminished responsiveness to sensory transmission, despite overall increases in excitability. Substance P, implicated in lung and NTS signaling, contributed to the increased responsiveness to current injections but not to the diminished sensory transmission. The finding that NTS neurons undergo plasticity with repeated ozone exposures may help to explain the adaptation of the respiratory motor responses.

membrane properties; brain stem; air pollutant


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE EXPOSURE TO HIGH AMBIENT levels of ozone evokes acute respiratory responses including airway inflammation, airway hyperreactivity, decreased forced vital capacity, cough, and rapid shallow breathing (59, 63). Infants and children are particularly vulnerable inasmuch as they exhibit greater incidences of adverse respiratory responses to ozone (63). In addition, exposing exercising children to high levels of ozone is thought to contribute to the development of asthma (45). The mechanism(s) for the ozone-induced respiratory responses is not completely understood, although it is known that ozone exposure causes local release of inflammatory mediators, oxidative stress, and activation of sensory neurons, all of which may contribute to the respiratory motor responses (11, 26, 59, 65). However, what may have been underappreciated is that the released inflammatory mediators, oxidative stress, and sensory nerve activation also trigger increases in central nervous system (CNS) neuronal activity that may further exacerbate the asthmalike symptoms (64). Two types of primary sensory nerves innervating the lungs, the vagal nonmyelinated lung C fibers and thinly myelinated Adelta rapidly adapting receptors, are activated by acute ozone exposure (11). When activated, the sensory endings initiate protective lung-CNS reflexes resembling the responses associated with acute ozone exposures: bronchoconstriction, airway hyperreactivity, cough, and rapid shallow breathing (11, 61).

A salient characteristic of ozone exposure that may relate to the underlying mechanism(s) of the respiratory responses is that with repeated daily or episodic ozone exposures the respiratory responses adapt. In general, the respiratory responses are enhanced on the second day of exposure, begin to wane on days 3 and 4, and become negligible by day 5 in humans (19, 24) and animals (61). In a previous study in rats with similar exposure protocol as the present study, all ventilatory parameters (breathing frequency and tidal volume) returned to preexposure levels by day 5 (57). In rats, the respiratory responses (rapid shallow breathing and decreased lung function) adapt despite the continued presence of lung epithelial damage, inflammation, increased concentrations of lavagable proteins (24), and increased sensory nerve excitability (30, 32), suggesting that lung-CNS reflexes might be involved in the adaptation. Of direct relevance to the present study is that the adaptation is not just limited to adults but also occurs earlier in life (37, 38).

To the extent that lung-CNS reflexes shape the respiratory responses to ozone exposures, neurons in the central network should exhibit changes in their behavior. Neurons in the nucleus tractus solitarius (NTS) are attractive targets for neurobehavioral changes inasmuch as it is at these synapses that the sensory information is integrated, modified, and ultimately relayed through divergent pathways to generate the reflex respiratory and pulmonary motor outputs (4, 43). CNS neurons in general have the capacity to undergo neuroplasticity in response to changes in patterns and frequency of synaptic inputs (8, 9, 51); moreover, this particular brain stem region lacks a complete blood-brain barrier (23), rendering the NTS neurons directly vulnerable to circulating inflammatory mediators released during ozone exposures. Whether and how repeated ozone exposures alter the behavior of these NTS neurons will shape the CNS neural contribution to the respiratory consequences of ozone exposures and may provide insights into the adaptation of the ozone-induced respiratory motor outputs.

Thus we asked whether extended episodic exposures to ozone (which is similar to real-life patterns of exposure to which humans are subjected) would change the neural behavior of neurons in the caudomedial NTS. The studies were performed in infant primates, a model that is morphologically, physiologically, and immunologically similar to human children. Whole cell recordings were made from neurons in transverse brain stem slices taken from ozone- and filtered air-exposed primates. Four properties were compared: 1) resting membrane potential; 2) resting input resistance; 3) excitability to nonspecific excitation evoked by depolarizing current injections; and 4) excitability to synaptic excitation evoked by stimulation of the tractus solitarius. Because substance P acting at neurokinin 1 (NK1) NTS receptors augments lung C-fiber reflex motor output (50) and because ozone exposure upregulates substance P in the airways (26), we further determined whether endogenous substance P in the NTS might contribute to potential ozone-induced changes in neuronal excitability.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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

Exposure Protocols

All 12 monkeys used for this study were California Regional Primate Research Center colony-born rhesus macaques (Macaca mulatta). Care and housing of animals before, during, and after treatment complied with the provisions of the Institute of Laboratory Animal Resources and conforms to practices established by the American Association for Accreditation of Laboratory Animal Care. Monkeys were randomly assigned to either a filtered-air control or ozone-exposed group (6 monkeys in each group). Ozone exposures started when the monkeys were 30 days old. The monkeys were exposed to 11 episodes of ozone (0.5 ppm for 8 h/day for 5 days followed by 9 days of filtered air). The studies were performed 3-5 days after the last exposure.

Brain Stem Slice Preparation

The monkeys were sedated with Telazol (8 mg/kg im) and killed with an overdose of pentobarbital sodium. After decapitation, the brains were rapidly exposed and submerged in ice-cold (<4°C) high-sucrose artificial cerebrospinal fluid (aCSF) that contained (in mM) 3 KCl, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 220 sucrose, and 2 CaCl2 (300 mosM), pH 7.4 when continuously bubbled with 95% O2-5% CO2. Brain stem coronal slices (250 µm thick) were cut with the Vibratome 1000 (Technical Products International, St. Louis, MO). After incubation for 45 min at 37°C in high-sucrose aCSF, the slices were placed in 37°C normal aCSF that contained (in mM) 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 25 NaHCO3, 25 glucose, and 2 CaCl2 (300 mosM), pH 7.4 when continuously bubbled with 95% O2-5% CO2. During the experiments, a single slice was transferred to the recording chamber, held in place with a silk mesh, and continuously perfused with oxygenated aCSF at a rate of ~3 ml/min. All experiments were performed at 33-34°C.

Whole-Cell Current-Clamp Recordings

Each slice was viewed by using a fixed-stage upright microscope equipped with infrared differential interference contrast for visualizing the neurons. Borosilicate glass electrodes were filled with a KCl solution containing (in mM) 140 KCl, 5 NaCl, 1 MgCl2, 3 K-ATP, 0.2 Na-GTP, 0.2 EGTA, and 10 HEPES (300 mosM), pH 7.4. Whole-cell recordings in NTS neurons were made with the Axoclamp 1D patch-clamp amplifier (Axon Instruments, Union City, CA). Whole cell voltages were filtered at 2 kHz, digitized at 10 kHz with the DigiData 1200 Interface (Axon Instruments), and stored in a 433 DX/D computer. The pipette resistance was 2.5-5.5 MOmega . The seal resistance was always greater than 1 GOmega , and the series resistance was no larger than 25 MOmega . Data were analyzed off-line by use of the pCLAMP6 software (Axon Instruments).

All experiments were performed on neurons located in the caudomedial NTS that displayed stable resting membrane potentials and that were activated by stimulation of sensory afferent fibers in the tractus solitarius, as evidenced by either an excitatory postsynaptic potential (EPSP) or a spike evoked by stimuli applied to the tractus solitarius ipsilateral to the recording site. Square-wave 0.1-ms pulses were delivered through bipolar tungsten electrodes (1-µm tips separated by 100 µm) to the tractus solitarius. Onset latencies for evoked spikes or EPSPs were determined from five successive stimuli applied to the tractus solitarius.

Effect of Ozone Exposures on Membrane Properties

The resting membrane potential was measured immediately after the whole-cell configuration. Then the voltage was current-clamped at -60 mV, so that all neurons were studied at the same membrane potential. Steady-state input resistance was determined by the slope of the relation between the injected hyperpolarizing currents (10-40 pA, 200-ms-duration pulses) and the resultant steady-state change in membrane potential.

Effect of Ozone Exposures on Neuronal Excitation

Neuronal responsiveness to nonspecific excitation was tested by injecting brief (2 s) intracellular depolarizing current pulses in 10-pA steps from 10 to 100 pA and measuring 1) total number of spikes evoked; 2) the maximum peak frequency of the evoked spikes; 3) spike frequency adaptation, defined as the ratio of the number of spikes in the second second to the number of spikes in the first second at 80 pA current injection; and 4) minimum current required to evoke a spike.

Neuronal responsiveness to synaptic excitation by sensory afferent fiber input was tested by stimulating the tractus solitarius and determining whether a spike was evoked at stimulating voltages starting at 2 V and increased to a maximum of 30 V.

Effect of Substance P (NK1) Receptors

The effect of substance P (NK1) receptors on the neuronal properties was tested with one of two substance P (NK1) receptor antagonists, CP-96,345 (10 µM) and SR-140,333 (1 µM). The input resistance, neuronal responsiveness to nonspecific excitation (depolarizing current injections), and neuronal responsiveness to synaptic activation by sensory fiber (tractus solitarius) stimulation were determined before and during perfusion of antagonist. The stimulation intensity to the tractus solitarius was adjusted so that each neuron responded with a spike ~40% of the time before the antagonist; the response rate during antagonist was tested with the same stimulation intensity. The effect of CP-96,345 on neuronal excitability was tested 3 min into the antagonist perfusion. Because the effect of SR-140,333 has a slower dissociation constant, the neuronal excitability was tested at least 10 min after the antagonist perfusion (17). Preliminary data analysis showed that there was no significant difference between the effect of CP-96,345 and SR 140,333, so the data were pooled.

Data Analysis

Data are expressed as means ± SE. Significance was set at P < 0.05. An unpaired t-test was used to compare the resting membrane potential, input resistance, the minimum injected current required to evoked a spike, spike-frequency adaptation, and the onset latency of the tractus solitarius-evoked spikes or EPSPs. A two-way ANOVA was used to determine the total number of spikes and peak spiking frequency evoked by the depolarizing current injections with exposure as the between factor and injected current as the within factor. For comparing the neuronal responsiveness to sensory afferent fiber excitation, the number of neurons that responded with a spike and the number of neurons that responded only with EPSPs at stimulating voltage less than 30 V were determined for each group and analyzed with chi 2. The lowest stimulating voltage required to evoke a spike was compared by using a Mann-Whitney rank sum test.

The effect of substance P (NK1) receptor antagonist on input resistance and the neuronal response rate to tractus solitarius input was analyzed with a two-way ANOVA with exposure as the between factor and in the absence and presence of antagonist as the within factor. The reduction in the number of spikes after antagonist perfusion (delta) at each injected current was analyzed with a two-way ANOVA with exposure as the between factor and injected current as the within factor.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Data were obtained from 52 neurons recorded in the NTS: 26 neurons each from the ozone- and filtered air-exposed group. Figure 1 shows an example the NTS recording configuration. The lower magnification microphotograph shows the position of the stimulation electrode in the tractus solitarius and the recording electrode in the NTS. The inset shows the recorded neuron in whole cell configuration under higher magnification. This neuron discharged an action potential to tractus solitarius stimulation as shown on the trace on the right. All neurons studied were located in the intermediate and caudal NTS from 1 mm rostral to 1 mm caudal to the obex and medial to the tractus solitarius. All neurons were activated with short-latency tractus-evoked spikes or EPSPs. There was no exposure-related difference in the onset latencies of the tractus solitarius-evoked spikes (4.2 ± 0.4 and 5.0 ± 0.6 ms for filtered air- and ozone-exposed monkeys, respectively; unpaired t-test: P = 0.266) and EPSPs (2.7 ± 0.2 and 2.9 ± 0.2 ms for filtered air- and ozone-exposed monkeys, respectively; unpaired t-test: P = 0.203).


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  		Fig. 1.   Recording configuration in the nucleus tractus solitarius (NTS) slices. Photomicrograph shows the position of the stimulating electrode in the tractus solitarius (TS) and the recording electrode in the NTS (bar, 100 µm). Inset: whole-cell formation of the recorded NTS neuron at higher magnification (bar, 10 µm). Right trace: action potential response of the neuron to tractus solitarius stimulation (black-down-triangle , stimulation artifact).

Effects of Exposures on NTS Neuron's Excitability

Intrinsic membrane properties. Extended repeated ozone exposures significantly depolarized the resting membrane potential of NTS neurons (Fig. 2A; unpaired t-test: P = 0.024). The more depolarized resting membrane potential was associated with a significant increase in input resistance (Fig. 2B; unpaired t-test: P = 0.006).


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  		Fig. 2.   Group data showing resting membrane potential (A) and input resistance (B) of neurons (number in parentheses) from filtered air control (FA) and ozone-exposed monkeys. Ozone exposure depolarized the resting membrane potential and increased the resting input resistance. *P < 0.05, unpaired t-test.

Neuronal responsiveness to depolarizing current injections. The ozone exposures also increased the neuronal excitability to depolarizing current injections. Figure 3A shows examples of the spiking responses of NTS neurons from an ozone- and filtered-air exposed monkey. The spiking responses of the two neurons were similar at the threshold depolarizing current injection (20 pA), but at the two higher depolarizing currents of 50 and 80 pA the neuron from the ozone-exposed monkey discharged more spikes than the filtered air-exposed control monkey. The group data (Fig. 3B) illustrate that the total number of spikes discharged by NTS neurons in response to increasing depolarizing current injections was significantly greater when the monkeys were exposed to ozone compared with filtered air (two-way ANOVA: P = 0.308, exposure; P < 0.001, current; P = 0.023, interaction). The increase in spiking was not accompanied by a higher instantaneous peak frequency of the spike discharge (two-way ANOVA: P = 0.673, exposure; P < 0.001, current; P = 0.949, interaction) or by a lower spike frequency adaptation in the ozone-exposed monkeys (ratio of spike discharge in the second second to that in the first second at 80 pA was 0.6 ± 0.1 and 0.7 ± 0.1 for filtered air- and ozone-exposed monkeys, respectively; unpaired t-test: P = 0.427). There was also no difference in the minimal depolarizing current required to evoke a spike (27 ± 4 and 24 ± 4 pA for filtered air- and ozone-exposed monkeys, respectively; unpaired t-test: P = 0.631).


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  		Fig. 3.   Neuronal response to intracellular depolarizing current (10-100 pA) injections in FA and ozone-exposed (Ozone) monkeys. A: examples of the spiking response to depolarizing current injections (20, 50, and 80 pA). I, injected current. B: group data showing the depolarizing current-evoked total number of spikes in FA- and ozone-exposed monkeys. As the amplitude of the depolarizing current injection was increased, the difference in the number of spikes evoked in the NTS neurons from the ozone-exposed monkeys became more prominent with respect to the FA monkeys. *P < 0.05, interaction, 2-way ANOVA.

Neuronal responsiveness to synaptic excitation by sensory afferent fiber stimulation. Ozone exposures decreased the overall neuronal responsiveness to sensory afferent input. As shown in Fig. 4A, at tractus solitarius stimulating voltages <= 30 V, 21 of 26 (81%) neurons from the filtered air-exposed monkeys discharged a spike compared with a significantly smaller 10 of 26 (38%) neurons from the ozone-exposed monkeys (chi 2 test: P = 0.005). In those neurons that responded with a spike at stimulating voltages <= 30 V, the mean stimulating voltage required to evoke a spike was significantly lower (Fig. 4B) in the filtered air-exposed group (13.7 ± 2.0 V) compared with the ozone-exposed group (21.6 ± 3.3 V, Mann-Whitney rank sum test: P = 0.033).


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  		Fig. 4.   Neuronal response to synaptic input. A: percent of neurons responding with a spike to stimulation voltages of <= 30V applied to the tractus solitarius in FA control and ozone-exposed groups. A significantly smaller percentage of neurons from the ozone-exposed infant monkeys responded with a spike compared with those in the FA-exposed group. *P < 0.05, chi 2. B: minimal stimulating voltage required to evoke a spike in the neurons responding to voltages <= 30 V was higher in the ozone-exposed monkeys. *P < 0.05, Mann-Whitney rank sum test.

Contribution of Substance P (NK1) Receptor Blockade

Twenty-four of the 52 neurons were tested with the substance P (NK1) receptor antagonists (12 neurons from each group). As shown in Fig. 5, substance P (NK1) receptor antagonists (CP-96,345 or SR-140,333) significantly reduced the input resistance. The magnitude of the reduction was not different in the two groups (Fig. 5, two-way ANOVA: P = 0.289, exposure; P =0.012, antagonist; P = 0.188, interaction).


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  		Fig. 5.   Group data showing the effect of substance P [neurokinin 1 (NK1)] receptor antagonist on the input resistance in neurons from FA and ozone-exposed monkeys. Substance P antagonists similarly significantly reduced the input resistance in both groups (P < 0.05, antagonist effect; P > 0.05 interaction, 2-way ANOVA). aCSF, artificial cerebrospinal fluid.

NK1 receptor antagonism had a significantly greater effect on the spiking responses to depolarizing current injections in the ozone-exposed monkeys. At lower depolarizing current injections (in which the spiking responses were not different between groups), the effect of antagonist was similar between groups. At higher depolarizing currents in which the spiking responses were higher in the neurons recorded in the ozone-exposed monkeys, the NK1 receptor blockade had a greater effect (Fig. 6, two-way ANOVA for number of spikes before and during NK1 antagonist: P = 0.104, exposure; P < 0.001, antagonist; P = 0.025, interaction). In a separate group of infant monkeys, we performed a time control for the spiking response to depolarizing current injections. There was no difference in the spiking response between the first and second trial (n = 5, two-way ANOVA: P = 0.945, time; P < 0.001, current; P = 0.138, interaction). The total number of spikes evoked by 10-100 pA of depolarizing currents were 0 ± 0, 0 ± 0, 2 ± 2, 8 ± 2, 15 ± 2, 21 ± 3, 34 ± 7, 36 ± 7, 56 ± 12, and 58 ± 17 for the first trial and 0 ± 0, 0 ± 0, 1 ± 1, 7 ± 3, 17 ± 7, 29 ± 11, 33 ± 12, 45 ± 17, 48 ± 17, and 48 ± 14 for the second trial.


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  		Fig. 6.   Group data showing the effect of substance P (NK1) receptor antagonists on spiking response to nonspecific excitation in neurons from FA (n = 12) and ozone-exposed (n = 12) monkeys. The reduction in the number of spikes during antagonist was greater in neurons from ozone-exposed than in FA-exposed monkeys (P < 0.05, antagonist effect) and more pronounced at higher injected currents (P < 0.025, interaction, 2-way ANOVA). As the amplitude of the depolarizing current injection was increased, the difference in the number of spikes evoked in the NTS neurons from the exposed monkeys became more prominent with respect to the FA control monkeys. *P < 0.05, 2-way ANOVA.

Blockade of NK1 receptors appeared to have no effect on the neuronal responses to tractus solitarius stimulation in either group. The neuronal response rates at the preset stimulation voltage that evoked a spike ~40% of the time for filtered air control and ozone group were 45 ± 7 and 35 ± 8% before the antagonist and 30 ± 10 and 34 ± 15% during antagonist perfusion (two-way ANOVA: P = 0.796, exposure; P = 0.247, antagonist; P = 0.314, interaction).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study provides electrophysiological evidence consistent with the hypothesis that repeatedly exposing infant primates to ozone results in neuroplasticity in the NTS. The NTS neurons exhibited an increased nonspecific excitability, as evidenced by a more depolarized resting membrane potential associated with an increased input resistance and an increased spiking response to intracellular injections of depolarizing currents. By contrast, the same neurons, as a group, were less responsive to synaptic activation by stimulation of sensory afferent fibers in the tractus solitarius. Endogenous substance P acting at NK1 receptors contributed to the ozone-induced increase in neuronal responsiveness to current injection-induced depolarization but appeared to play no role in the ozone-induced diminution of the neuronal responsiveness to synaptic transmission between the sensory afferent fibers and NTS neurons. All neurons were located in the caudomedial NTS where vagal afferent fibers originating from the lungs and airways terminate (5) and were activated with short latencies to stimulation of the tractus solitarius, suggesting that they were part of autonomic, including pulmonary, afferent pathways.

With the more depolarized resting membrane potential shifting the neuron closer to the threshold for discharging action potentials, a subthreshold input, which might otherwise have no effect, could evoke postsynaptic spikes and hence a neuronal output. A suprathreshold input might evoke more spikes, amplifying the neuronal output. The resting membrane depolarization was associated with a higher membrane resistance (lower membrane conductance), suggesting the possible contribution of potassium conductance to the more depolarized membrane potential (7). The increased number of spikes evoked by depolarizing current injections suggests a heightened responsiveness to rapid changes in the membrane potential. Moreover, as the magnitude of the depolarizing current was increased, the effect of the ozone exposures became more prominent, suggesting that larger excitatory inputs would evoke disproportionately larger responses of NTS neurons after ozone exposures.

Despite the generalized increase in excitability to membrane depolarization, the NTS neurons were less likely to be synaptically activated by sensory afferent input from vagal afferent fibers in the tractus solitarius, suggesting that the ozone exposures may have specifically decreased the likelihood of transmission of lung-related sensory information to the NTS neurons. Given that ozone exposure causes local release of inflammatory mediators, oxidative stress, and activation of sensory neurons (11), it is possible that the extended exposure to ozone could have damaged the sensory afferent fibers through one of these mechanisms, perhaps decreasing the number of synapses to the NTS and thereby decreasing the likelihood that activation of the tractus solitarius would have evoked synaptic responses.

It is also tempting to speculate that this decreased likelihood of synaptic transmission might help to explain the phenomenon of adaptation of the respiratory responses that occurs with repeated or episodic ozone exposures. Several mechanisms have been proposed to mediate the adaptation, including increased cellular antioxidants (16, 55), changes in lung cell populations to cells less sensitive to ozone (25, 48), changes in mucus or surfactant properties (14), and changes in cytokine signaling (46). The decreased capacity of the neurons to be synaptically activated by peripheral sensory input might provide an additional mechanism, particularly in view of the findings that adaptation of the reflex responses occurs despite persistent morphological and biochemical changes in the lung (that might be expected to provide a continued stimulation of the sensory nerve fibers) and persistent increases in excitability of the rapidly adapting receptor sensory nerve afferent fibers (30, 32) that might be expected to provide an increased level of afferent traffic to the NTS. The ultimate result of this adaptation may be to leave the lung more vulnerable to injury because the reflexes are thought to be protective (10). In that regard, we have shown that a rapid shallow breathing pattern as occurs with stimulation of lung C fibers, in fact, reduces ozone-induced epithelial injury in isolated lungs (31).

Although no studies have investigated ozone-induced changes in neuronal behavior in the NTS, several animal studies have provided evidence of neurobehavioral plasticity after ozone exposure throughout the CNS, resulting in memory deficits (56), alterations in cortical visual evoked potentials (3), disruptions of sleep patterns (53), generation of social and aggressive behavior (49, 54), and subtle changes in swimming navigation, an indicator of hippocampal dysfunction. These neurobehavioral changes have been linked to both structural plasticity, including changes in dendritic spine densities and neurochemical plasticity including decreases in catecholamine (12) and serotonin levels (2), as well oxidative stress (56). Studies in humans have also reported CNS effects, including impaired vigilance levels, decreased physical performance, headache, and lethargy (20, 22). In the context of the known effects of ozone in the CNS, the present findings indicate that, depending on the function of the neuronal network, ozone effects may be manifest not only as cognitive or somatic motor changes, but also as changes in respiratory motor output.

Because substance P has been implicated in environmental exposures (18, 41) and in lung CNS reflexes, we examined the potential role of NK1 receptors in the ozone-induced neuroplasticity. The neuropeptide is synthesized in the cell bodies of vagal afferent fibers (21, 28) and in the NTS (13). Substance P synthesized in the vagal cell bodies can be transported distally and released in the airways as well as being transported centrally where it can be released in the NTS (27, 29). The substance P content in the airways increases after exposure to other environmental pollutants such as tobacco smoke (41) and allergens (18), and its release is increased after ozone exposure in humans (26). Substance P-containing nerve terminals are heavily distributed in the NTS (28, 36, 60), and substance P activation of NK1 receptors on NTS neurons has been shown to augment lung C-fiber nerve reflex output (50). In the present study, the finding that NK1 receptor antagonism suppressed the spiking responses to depolarizing current injections to a significantly greater extent in the ozone-exposed monkeys compared with the filtered air-exposed controls suggests that upregulated substance P mechanisms may contribute to the ozone-induced increase in postsynaptic excitability. If ozone somehow damaged the substance P-containing sensory afferent fibers, it could be possible that a consequence was an upregulation of NK1 receptors on the NTS neurons. Our laboratory has shown (33) that injuring C-fibers with high doses of capsaicin increased the affinity of substance P for NK1 receptors. So, on depolarization, the patch-clamped neuron released an excitatory neurotransmitter onto intermingled substance P-containing interneurons, which, in turn, released substance P to activate (the upregulated) NK1 receptors on the patch-clamped neuron. Even with this speculation, however, whether the ozone-induced increases in the substance P effects were due to changes in substance P levels and/or in elements of the NK1 receptor system has not yet been determined. Despite the substance P effects on the spiking responses, there was no contribution to the decreased likelihood of synaptic activation. Because we were able to detect statistically significant differences in the contribution of substance P to the spiking responses, we assume that significant differences in the contribution of substance P could have been detected for the synaptic responsiveness. Still, given that those findings were negative, we cannot be certain as to whether a larger sample size could have revealed a significant effect of the substance P antagonist.

Regarding the selectivity of the antagonists used in the present study, the same or higher concentrations of SR140,333 have previously been used for selective NK1 receptor antagonism in sympathetic neurons (34), parasympathetic ganglion neurons (35), human isolated bronchi (1), and mouse hippocampal slices (39, 40). Up to 1 µM of SR140,333 has been shown to have no effect in bioassays for NK2 ([beta Ala8]neurokinin A-induced contraction of endothelium-deprived rabbit pulmonary artery) and NK3 ([MePhe7]neurokinin B-induced contraction of rat portal vein) receptors (17); however, in expressed Chinese hamster ovary cells (52) the antagonist did block NK3 receptors at high doses, raising the possibility that NK3 receptors could also have been affected in this study. On balance, however, it seems reasonable to conclude that the antagonist effects seen with SR-140,333 were most likely mediated by NK1 receptor antagonism. CP-96,345 has been shown to interact with Na+ and Ca2+ channels (6, 58). Lepre et al. (42) showed that, although having no effect on membrane potential, 10 µM of CP-96,345 shortened the slow afterhyperpolarizations (presumably through the nonspecific action on voltage-dependent L-type Ca2+ channels) and induced repetitive firing on the spiking response to intracellular depolarizing current injections (possibly secondary to the shortened slow AHP) in spinal motoneurons. However, given that CP-96,345 reduced the spiking response in the present study (rather than increasing spiking response as in the study by Lepre et al.) and that the effects of CP-96,345 and SR-140,333 were similar, it seems likely that the antagonist effects were largely NK1 receptor mediated.

The studies were performed in infant primates, an ideal model for investigating mechanisms underlying ozone-induced effects in children, given the morphological, physiological, neuroanatomical, and immunological similarities. Although there are limitations, as with any study using nonhuman primates (e.g., the availability of animals and tissues due to the costs and thus the small sample sizes), the advantages of studying neural processing in the nonhuman primate as a model of human neuronal behavior far outweigh the limitations. Despite the small sample size, by using whole cell recordings in brain stem slices the study was able to detect statistically significant differences in two out of three intrinsic membrane properties of neurons taken from the filtered air- and ozone-exposed monkeys. In addition, although all recordings were made in the same NTS region where lung sensory afferent fibers terminate and all neurons were shown to receive sensory afferent input via the tractus solitarius, owing to the need to coordinate these neurophysiological studies with a number of studies on various other aspects of the consequences of ozone exposures in these monkeys, it was not possible to inject dye in the lung to confirm pulmonary input to the NTS neurons. Still, given the localization of lung sensory afferent fiber synapses in this NTS region, it seems reasonable to assume that most of those neurons studied have some relationship to lung reflexes and hence respiratory motor output.

The use of the in vitro slice preparation for studying neuroplasticity is well established, particularly in hippocampal slices, in which long- and short-term plasticity has formed the basis for cellular mechanisms underlying learning and memory (44, 51). The advantages of the preparation, the simplicity, mechanical stability, and ability to study intrinsic membrane properties and synaptic currents are appreciated, but there are limitations that must also be recognized, such as loss of the entire reflex circuitry of the whole animal, and concerns about oxygenation and health of the slice. In the present study, all neurons studied were with 20 µm of the surface interfacing with the oxygenated perfusate, and in whole brain stem preparations the PO2 and pH gradients have been shown to remained virtually constant for the first 100-200 µm before decreasing in a curvilinear fashion (62), suggesting that the effects we attributed to ozone were not secondary to poor oxygenation of the slice. Finally, the resting membrane potential and input resistance were consistent with those observed numerous other studies (15, 47), suggesting that, notwithstanding the limitations of in vitro preparations, the neurons were healthy and able to discharge action potentials.

In summary, these findings demonstrate that exposing infant primates episodically to ozone caused neuronal behavioral changes in a CNS region that contains neurons that process lung sensory signals. These NTS neurons displayed a reduction in synaptic transmission despite increases in generalized excitability that was mediated in part via a substance P (NK1) receptor mechanism. The physiological relevance of these findings may relate to the phenomenon of adaptation of the reflex respiratory motor responses.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the contributions of the Respiratory Diseases Unit at the California Regional Primate Research Center, the support of Primate Services at the California Regional Primate Research Center for animal handling, care, and coordination and veterinary care. SR-140,333 was a generous gift of Sanofi Recherche, Montpellier, France.


    FOOTNOTES

This work was funded by National Institute of Environmental Health Sciences Grant P01 ES-00628.

Address for reprint requests and other correspondence: A. C. Bonham, Univ. of California at Davis, Dept. of Pharmacology & Toxicology, Tupper Hall 1228, One Shields Ave., Davis, CA 95616 (acbonham{at}ucdavis.edu).

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.

First published October 25, 2002;10.1152/japplphysiol.00552.2002

Received 25 June 2002; accepted in final form 16 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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