Salicylate is the most commonly used drug for relief from inflammation, pain, and fever. Unfortunately, suicide by intentional salicylate overdose accounts for a substantial number of deaths. Artificial ventilation is required for support of poisoned patients. The effects of salicylate on respiration contribute to the serious acid-base balance disturbances that characterize poisoning by this class of compounds. Full therapeutic doses of salicylate increase oxygen consumption and CO2 production. These effects are a result of salicylate-induced uncoupling of oxidative phosphorylation (1). Salicylate directly affects the respiratory center in the medulla (2–5).
In the brainstem, the rostral ventrolateral medulla (RVLM) is thought to be an important area for respiratory rhythm generation (6) and may be the target area for salicylate-induced respiratory stimulation and depression. Respiratory neurons in the brainstem-spinal cord preparation occur in three major classes according to the pattern of membrane potential oscillations and spike discharges: inspiratory, preinspiratory, and expiratory neurons (6,7). Inspiratory neurons are the most important respiratory neurons in the RVLM because they comprise the final motor output of medullary respiratory neurons (8). However, the effects of salicylate on membrane properties of medullary inspiratory neurons of the ventral respiratory group in the RVLM have not yet been established. We hypothesized that salicylate has a direct action on the respiratory center and that different concentrations of salicylate have different actions. The purpose of this study was to examine salicylate effects on medullary inspiratory neurons in a brainstem-spinal cord preparation from newborn rats.
This study was approved by the Animal Care and Use Committee of Nippon Medical School. Brainstem-spinal cord preparations from 0- to 4-day-old Sprague-Dawley rats (16 preparations) were used (6,9). Brainstem and spinal cord were isolated under deep ether anesthesia (10). The brainstem was rostrally decerebrated between the sixth cranial nerve roots and the lower border of the trapezoid body. Most of the pons was removed. The preparation was then placed in a perfusion chamber (2 mL) with its ventral surface up and was continuously superfused at 3.0–3.5 mL/min with artificial cerebrospinal fluid (aCSF) (124 mM NaCl, 5.0 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, and 30 mM glucose), equilibrated with 95% oxygen and 5% CO2, and maintained at 26°–27°C and a pH of 7.4.
Intracellular whole-cell recordings were made from the inspiratory neurons in the RVLM by using a blind patch-clamp technique (7,11). Electrodes were pulled from thin-walled borosilicate glass capillary tubing containing a filament (GC100TF-10, outer diameter 1.0 mm; Clark Electromedical, Reading, UK) on a vertical puller. The electrodes had an inner-tip diameter of 1.2–2.0 μm and a direct current resistance of 3–8 MΩ. The electrode solution consisted of 130 mM potassium gluconate, 10 mM EGTA, 10 mM HEPES, 2 mM Na2-adenosine triphosphate, 1 mM CaCl2, and 1 mM MgCl2, pH 7.2–7.3, adjusted with KOH. The electrodes were placed in the RVLM, including the ventral respiratory group, by insertion through a small region of the ventral surface of the RVLM where the pia mater had been removed with a thin glass needle. To keep the tip of the patch electrode clean, slight positive pressure (10–30 mm Hg) was applied. Neurons were sought by advancing the electrode into the RVLM while monitoring amplified extracellular signals with an audio amplifier monitor. When the electrode approached an inspiratory neuron, which was functionally identified by monitoring extracellular action potential discharges, an increase in the voltage response of the patch electrode to injection of negative direct current, by up to 150%, was observed. Subsequently, positive pressure was released and negative pressure was applied for a >1-GΩ seal formation. Afterwards, the whole-cell configuration was established by gentle suction, which was, in some cases, combined with injection of a single-shot hyperpolarizing current pulse (amplitude, 0.6–1.0 nA; duration, 30 ms). Membrane potential (mV) was recorded with a single-electrode voltage clamp amplifier (AxoClamp 2B; Axon Instruments, Foster, CA) after compensation for the series resistance (20–60 MΩ) and capacitance. Discharges of respiratory motor activity were recorded extracellularly with suction electrodes applied to the proximal ends of cut ventral roots of spinal (C4 or C5) nerves. Signals were fed through a high-pass filter with a 0.3-s time constant. During the experiments, neuronal activity was displayed on a chart recorder, monitored with an oscilloscope, digitized (Digidata 1200B; Axon Instruments), and stored on a hard disk for off-line analysis with a personal computer and data acquisition software (Axoscope; Axon Instruments).
aCSF was applied for 10 min as control. aCSF containing 1 or 10 mM salicylate (Sigma, St. Louis, MO) was then applied for 10 min. This was followed by application of the γ-aminobutyric acid (GABA) receptor antagonist bicuculline (Sigma; 1 μM) for 10 min. All solutions were adjusted to a pH of 7.4 and were applied to the recording chamber through the perfusion system. Each preparation was exposed once to a single concentration of salicylate.
Repeated-measurement analysis of variance and Bonferroni tests were performed to distinguish differences over time. Statistical analyses were performed with SPSS version 8.0J (SPSS Inc., Chicago, IL). All values are reported as mean ± sd, and all P values <0.05 were considered to be statistically significant.
Application of 1 mM salicylate (n = 8) resulted in a synchronous increase in the burst rate of inspiratory neurons and of the C4 activity from 6.9 ± 1.6 bursts/min to 8.2 ± 1.9 bursts/min (P < 0.05;Figs. 1 and 2). The burst rate of inspiratory neurons and the C4 activity were decreased from 8.3 ± 0.7 bursts/min to 4.5 ± 1.1 bursts/min after application of 10 mM salicylate (n = 8) (P < 0.01;Figs. 1 and 3). The depressant effects of 10 mM salicylate were antagonized by 1 μM bicuculline, whereas the increase in the burst rate of inspiratory neurons and of C4 activity caused by 1 mM salicylate was not changed by 1 μM bicuculline. Resting membrane potential (48.6 ± 3.6 mV) and intraburst firing frequency in the inspiratory neurons did not change on application of salicylate and bicuculline (Table 1). When the depressant effects of 10 mM salicylate were antagonized by 1 μM bicuculline, the burst duration of inspiratory neurons decreased significantly (Table 1).
The major findings of this investigation were that small-dose (1 mM) salicylate results in an increase in the burst rate of inspiratory neurons, whereas a large dose of salicylate, at toxic levels (10 mM), inhibits inspiratory neuron bursts. The GABA receptor antagonist bicuculline reverses the inhibition of inspiratory neurons by large-dose salicylate. The actions of salicylate on medullary inspiratory neurons appear to be primarily caused by a presynaptic effect, but a postsynaptic effect is thought to also be partly responsible.
The brainstem-spinal cord preparation from newborn rats preserves a basic neuron network and the function of the respiratory center in the medulla (6,9,11). This in vitro preparation, maintained under anesthesia-free conditions, is suitable for detailed pharmacological studies of the respiratory center because drugs can be applied at defined concentrations into regions of interest by superfusion (9). Whole-cell patch-clamp recordings make it possible to analyze the electrophysiological properties of respiratory neurons in this preparation (6–9,11). In this study, central mechanisms of respiratory depression induced by salicylate were investigated by using the brainstem-spinal cord preparation from newborn rats described above. Although this reduced preparation presents technical advantages, extrapolation of results to the mature and intact animal should always be made with a degree of caution.
Salicylate increases metabolic rate and CO2 production through its ability to uncouple oxidative phosphorylation (1). Salicylate is also believed to have a direct effect on respiratory neurons in the medulla (2–5). This direct effect is suggested by findings of profound respiratory alkalosis with salicylate toxicity and has been further substantiated in animal studies by the central application of salicylate (3,4). It has been reported that CSF acidosis is not the central mechanism of salicylate-induced hyperventilation (3). Moreover, it has also been shown that salicylate has no effect on the CSF H+ concentration in dogs (5). The central action of salicylate is much more important for increasing ventilation than for effects related to uncoupling of oxidative phosphorylation (4). Intracisternal administration of salicylate causes hyperventilation without an increase in whole-body metabolism, suggesting that salicylate may act directly or through mediators on neurons involved in the control of breathing (2). Furthermore, although most of the therapeutic effects of salicylate are due to inhibition of the cyclooxygenase system, prostaglandin inhibition does not appear to be the mechanism of salicylate-induced central hyperventilation (12). Salicylate action on the central respiratory system does not involve inhibition of prostaglandin synthesis or alteration of the central nervous system acid-base balance (5,12). The site of action of salicylate is most likely one or more of the chemosensitive areas on the ventrolateral medullary surface (13,14). It has been shown that the central ventilatory effects of salicylate act through cholinergic mechanisms (15).
These results, showing a stimulus effect of salicylate on respiration, are consistent with those of previous studies. The serum salicylate concentration at which detectable changes in ventilation occur is 13.7–22.4 mg/dL (1–1.6 mM) (16). An increase in minute ventilation from 6 to 7 L/min occurs with mean serum salicylate concentrations of 18.6 mg/dL (1.3 mM) (17). Salicylate-induced hyperventilation reduces the frequency and duration of sleep apnea at a serum level of 33.3 mg/dL (2.4 mM) (1). The salicylate concentration used in this work (1 mM) was enough to cause an increase in the inspiratory neuron burst rate. Therefore, salicylate directly stimulates the respiratory center in the medulla.
A depressant effect of salicylate on the medulla appears after large-dose exposure. Toxic doses of salicylate cause central respiratory paralysis (18). Salicylate concentrations larger than 6.5 mM are extremely toxic (19). Large-dose salicylate (10 mM) caused central respiratory depression in this study by inhibiting medullary inspiratory neurons. This depressant effect of salicylate was completely antagonized by bicuculline. GABA-like inhibition is therefore likely to be involved in the periodic inhibition of medullary respiratory neurons (20,21). However, inspiratory neurons in the RVLM and, probably, the inspiratory neurons composing the inspiratory pattern generator, have few GABA receptors (22). The effects of bicuculline on inspiratory neurons might be produced by disinhibition of excitatory inputs rather than by direct action (22). GABAergic mechanisms are most probably involved in the central inhibition of inspiratory neurons by salicylate.
When salicylate-induced respiratory depression was reversed by bicuculline, we found inspiratory neuron burst duration to be reduced significantly. Bicuculline increases the frequency and amplitude of inspiratory bursts (23). Inspiratory neuron burst duration tended to decrease during treatment with bicuculline (22). However, small-dose salicylate caused an increase in inspiratory neuron burst rate and a decrease in burst duration. The burst duration may be affected by the burst rate itself, with a shorter burst duration appearing at a faster burst rate (9). Our findings are partly consistent with this suggestion. Salicylate’s direct action on inspiratory neurons may play a role in shortening the inspiratory burst duration. Postsynaptic action, independent of the burst rate, of salicylate to inspiratory neurons may be involved in the decrease in burst duration.
The patterns of burst activity and the postsynaptic potentials of three major respiratory neurons in the RVLM suggest a complex pattern of mutual synaptic connections (9). Inspiratory neurons receive excitatory and inhibitory synaptic connections from preinspiratory neurons, reflected in the central respiratory rhythm, and also have inhibitory synaptic connections to preinspiratory neurons (9). In previous studies using the brainstem-spinal cord preparation from neonatal rats, it was suggested that bath-applied neuromodulators induced changes in respiratory rhythm, primarily through altering preinspiratory neuron activity (6,9). In this study, resting membrane potential did not change during the increase in inspiratory neuron burst rate with the application of 1 mM salicylate. Inspiratory neurons inhibited with 10 mM salicylate also had an unchanged resting membrane potential. In addition, the intraburst firing frequency of the inspiratory neurons did not change when the burst rate changed. This result suggests that the main action of salicylate on inspiratory neurons is presynaptic stimulation or inhibition of the excitatory synaptic inputs. This action is in contrast with the action of opioids (8). The main action of opioids is suggested to be on inspiratory neurons, and they do not affect the central rhythm effected by preinspiratory neurons. Salicylate, on the other hand, appears to cause a change in respiratory rhythm by its action on the central rhythm generator.
We conclude that small-dose salicylate directly stimulates and large-dose salicylate depresses the respiratory center in the brainstem-spinal cord preparation from newborn rats. The actions of salicylate on medullary inspiratory neurons seem to be primarily caused by a presynaptic effect; also, a postsynaptic effect is thought to be partly responsible. GABAergic mechanisms are probably involved in the salicylate-induced central respiratory depression.
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