Main

Adenosine has a number of physiologic effects [for reviews, see Berne(1) and Collis and Hourani(2)], including depression of respiration in fetal(3, 4), newborn(5, 6), and adult(7, 8) mammals. Adenosine is a constituent of ATP and is mainly formed by dephosphorylation of AMP. The breakdown of ATP during hypoxia (i.e. decrease in Po2) and the concomitant increase in adenosine levels in the brain(9) may contribute to the irregular breathing or apnea that is often seen both in preterm human infants and in preterm animal models(10, 11).

In the human neonate, apnea can be triggered by hypoxia(12, 13), whereas the adenosine antagonist 1,3,-dimethylxanthine (theophylline) blocks hypoxia-induced depression of breathing in rats(14), rabbits(6), and piglets(15, 16). Thus, the therapeutic effect of theophylline on neonatal apnea has been suggested to be due to its antagonistic action on adenosine receptors(17–19).

However, little is yet known about the site(s) at which adenosinergic inhibition of breathing occurs. Adenosine has been shown to inhibit excitatory postsynaptic currents in hippocampal neurons(20) and to decrease neurotransmitter release in prejunctional motoneurons(21). In addition to its inhibitory effect on central neurons, adenosine also decreases body temperature and oxygen consumption, which could indirectly decrease respiration(22). Thus, the extent to which adenosine influences breathing by decreasing body metabolism or by a direct action on central respiratory control mechanisms is not yet clear.

The involvement of adenosine in the depression of inspiratory depth, via modulation of synaptic transmission at the spinal level to phrenic motoneurons, has recently been demonstrated(23). However, the possible effects of adenosine on the RR in the neonate has not been determined. Nor has the possibility that the age dependency of the effects of adenosine on respiration, demonstrated in vivo(5), influences the central respiratory control mechanisms been examined.

In the present study we have characterized the effects of adenosine on central respiratory pattern generation. Subsequently, we examined the possible effects of adenosine on C4/C5 RR, inspiratory time and amplitude, and the firing rate of medullary neurons, as well as the possible dependency of these effects on the postnatal age.

To study the effects of adenosine on central respiratory control mechanisms during the neonatal period, we have used an in vitro neonatal rat brainstem-spinal cord preparation from newborn to 4-d-old pups. This preparation retains the functional circuits required for generating a complex, coordinated respiratory motor output of brainstem origin, resembling the respiratory pattern of vagotomized rats in vivo(24, 25). The brainstem-spinal cord preparation thus contains the neurons critical for rhythm generation and integration of respiratory drive, which have been demonstrated in vitro to be located in the rostral ventrolateral medulla(26), that also seems to contain vital respiratory neurons in vivo(27). Inspiratory neurons, which appear to be fundamental components of the inspiratory pattern generation, are found near the nucleus ambiguus, in a region corresponding to the rostral ventrolateral medulla in this brainstem-spinal cord preparation(28). We have thus focused our investigation of respiratory pattern modulation by adenosine on these neurons.

METHODS

Brainstem-spinal cord preparation. The experiments were performed on newborn (0-4-d-old) Sprague-Dawley rats (n = 62). The brainstem and spinal cord were dissected under deep ether anesthesia and isolated as described previously(24, 29). In short, the brainstem was rostrally decerebrated between the VIth cranial nerve roots and the lower border of the trapezoid body.

The preparation was then transferred to a 2-mL chamber and continuously perfused at a rate of 3.0-3.5 mL/min with the following standard solution(mM): NaCl 124; KCl 5.0; KH2PO4 1.2; CaCl2 2.4; MgSO4 1.3; NaHCO3 26; glucose 30; and equilibrated with 95% O2 and 5% CO2 to pH 7.4 at 27.5 °C. The pH (membrane pH-m, HI 8314, Hanna Instruments, Leighton Buzzard, Bedfordshire, UK) and the temperature of the bathing solution were continuously monitored. Temperature was measured either directly in the bath (Quartz Digi-Thermo) or indirectly in the water-heating bath (Julabo UC, Julabo 5B), after calibration by direct measurement.

Recordings. Discharges of spinal motoneurons were recorded with suction electrodes applied to the proximal ends of cut C4 or C5 ventral root containing respiratory motoneuron axons (Fig. 1). This C4/C5 activity corresponds to phrenic nerve discharges(24, 25). The C4/C5 activity was amplified, rectified, and further processed (integrated) by a third order (Paynter) R-C filter (time constant 100 ms), to obtain signals approximating a moving average of the recorded activity (Int. C4).

Figure 1
figure 1

The brainstem-spinal cord preparation. Schematic diagram of the neonatal rat brainstem-spinal cord preparation ventral surface. Respiration-related neurons were recorded extracellularly using a glass microelectrode. Respiratory motoneuron activity corresponding to inspiration was recorded from the C4/C5 ventral root through a glass suction electrode. Recordings to the right show spontaneous discharges from a Inspiratory neuron(top), C4 and the integrated C4 (bottom).Ttot = respiratory burst cycle, fi = intraburst firing frequency of I neuron, Ti = inspiratory time, I = duration of I neuron bursting, AICA = anterior inferior cerebellar artery, and XII = n. hypoglossus.

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Extracellular recordings of the unit activity of respiration-related neurons in the ventrolateral medulla were performed using a glass microelectrode filled with 2% Pontamine sky blue in 0.5 M sodium acetate(resistance 2-8 megohms). The electrode was inserted through the ventral surface into the left or right rostral ventrolateral medulla using a micromanipulator (MW-4, Narishige, Tokyo). Respiration-related neurons were found 50-600 μm from the ventral surface(28) and identified by their characteristic firing pattern and correlation in time to the respiratory cycle of C4/C5 activity. Neuronal units that discharged only during the inspiratory phase (I neurons) were included in this study. Signals were amplified and band pass-filtered (10 Hz to 5 kHz, differential AC amplifier model 1700, A-M Systems, Everett, WA).

The C4/C5 and I neuronal activities were monitored via an analogue-digital converter (Digidata 1200, Axon Instruments, Foster City, CA) and data acquisition software (Axotape, Axon Instruments). Data were sampled (0.3-3 kHz) and stored for 1-4-min intervals every 10 min on a computer for off-line analysis. Continuous recordings of signals were performed with a strip chart recorder (ABB SE 120).

Drugs and chemicals. Adenosine is rapidly metabolized and transported from the extracellular space(30). We therefore used the metabolically stable adenosine A1 receptor agonist R-PIA to examine the effects of adenosine on respiratory activity. A solution of R-PIA (RBI, Natick, MA) was made by dissolving 4.0 mg in 0.10 mL of DMSO and 3.90 mL of standard solution to give a stock solution of 1000 μg/mL. This solution was further diluted in standard solution to yield final concentrations of 0.1, 1.0, and 10.0 μM.

Dipyridamole, a nucleoside transport inhibitor, was used in some preparations to increase the endogenous adenosine levels. Dipyridamole (RBI, Natick, MA) was prepared in the same way as R-PIA to give a concentration of 50 μM. The final concentration of DMSO in the solutions was less than 0.01%.

Theophylline, the clinically used adenosine receptor antagonist, with an A1/A2 receptor selectivity of 3:1(31), and the highly specific A1 receptor antagonist DPCPX were also used to establish the type of adenosine receptors involved. Theophylline (Sigma Chemical Co., Sweden) was used in a concentration of 10-20 mg/L (55-110μM). A 1 mM stock solution of DPCPX (Sigma) in ethanol was diluted in standard solution to a final concentration of 200 nM. In control experiments, corresponding amounts of DMSO or ethanol added alone to the standard solution did not change the respiratory activity. Drugs were added to the standard solution superfusing the preparation after an initial control period of 100± 6 min.

Data analysis. The inspiratory time (Ti) was defined as the interval during which a continuos discharge occurred in the ventral root. The respiratory cycle duration in seconds(Ttot), defined as the time between two consecutive bursts of Int. C4, was used to calculate the RR in respiratory bursts per min (Fig. 1). As an index of the regularity of the respiratory pattern, the CV, i.e. the ratio of the SD and the mean cycle duration, was calculated.

The Int. C4 was analyzed with regard to change in amplitude. In some experiments (n = 20), the mean fi of I neurons was calculated from the average number of spikes in 10 consecutive bursts (Fig. 1). The medullary respiration-related neuronal response and the C4 motoneuronal burst activity were analyzed under control conditions, during drug application; after 21 + 0.5 min of perfusion with R-PIA or theophylline or 26 ± 0.5 min of perfusion with dipyridamole; and after recovery with the standard solution.

The mean and SEM were calculated for all data, and the results are listed as an average percentage of the control values ± SEM (% ± SEM).

Statistical significance was assessed using two-way analysis of the variance, repeated measures design to compare the activity before, during, and after drug application. The difference between two means was analyzed using t test after the F test was performed. The difference between several independent means was analyzed by comparing all pairs using the Tukey-Kramer HSD test. A value of p < 0.05 was considered to be statistically significant.

RESULTS

Control respiratory activity during development. A representative recording from an inspiratory neuron and the concomitant C4 activity of a 1-d-old rat are shown in Figure 2A. The spontaneous activity of the inspiratory-related neurons in the rostral ventrolateral medulla and the respiratory bursts recorded from C4/C5 remained stable for more than 4 h under control conditions. In preparations from pups with postnatal age >2 h, the control RR was 9.15 + 0.3 bursts/min with regular C4/C5 respiratory rhythm, the CV being 0.18 ± 0.02 and the Ti 784 ± 35 ms (n = 56). Control respiratory parameters in the preparations from different age groups are presented in Table 1 and Figure 3C. A more irregular breathing pattern (CV = 0.37 ± 0.08) was seen in all(n = 5) the preparations from newborn pups (0-2 h old)(Fig. 3, A and C). This pattern was characterized by respiratory bursts appearing in clusters of two or even three with short periods (e.g. <500 ms) between bursts. This aberrant pattern was not modified to a more regular one, even after sectioning the medulla more caudallly (n = 3), in an attempt to exclude possible remaining pontine inhibition(25). These three (0-2 h) preparations were not included in the analysis of drug effects.

Figure 2
figure 2

Effects of adenosine agonist. The effects of R-PIA and theophylline on inspiratory neurons (I neuron, upper traces), C4 activity (middle traces), and integrated C4 activity (lower traces) are shown. (A) The respiratory activities recorded with a preparation from a 1-d-old rat in standard solution. (B) As in(A), except after 21 min of perfusion with 1 μM R-PIA.(C) Washout 20 min after the R-PIA perfusion. (D) After 20 min of perfusion with 1 μM R-PIA and 55 μM theophylline.

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Table 1 Control respiratory activity in relation to postnatal age
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Figure 3
figure 3

The postnatal respiratory pattern. In the preparations made from pups within 2 h after birth, a pattern with respiratory bursts appearing in clusters was present. (A) Control respiratory C4 activity (C4) and Int. C4 of a preparation from a pup killed and dissected immediately after birth. (B) As in (A), except after 22 min of perfusion with 50 μM dipyridamole. (C) The regularity of the C4/C5 burst rhythm in relation to postnatal age. The CV of RR was significantly correlated to postnatal age (age vs CV; Spearman rank correlation p = 0.01) (P = postnatal age in days).

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A similar irregular respiratory pattern (CV = 0.49 ± 0.09), significantly different from the normal control, was seen in 3 out of 12 preparations from the slightly older pups (2-24 h), but only in one (27 h; CV= 0.56) of 45 preparations from pups older than 24 h. In total, nine preparations demonstrated irregular RR. Of these, three were excluded from analysis of drug effect, after the caudal sectioning of the medulla, mentioned above, had been performed. The remaining six preparations with irregular control respiratory patterns did not show any significant differences in drug effects compared with the preparations with more regular respiratory activity.

Preliminary analysis by Spearman rank correlation revealed a significant age dependency with respect to the effects of R-PIA and dipyridamole (see Fig. 5A) When the data from the preparations were divided into groups with postnatal ages of 0-2 h, 3-24 h, 25-47 h (P1), 48-72 h (P2) and >72 h (P3), the RR was significantly higher in the youngest group of pups (0-2 h). The Ti was not significantly different between groups (Table 1), but the regularity of the respiratory pattern (CV) differed significantly between the youngest (0-2 h) group and the P2 and P3 groups (Fig. 3C).

Figure 5
figure 5

Age dependency of adenosinergic effects on respiration.(A) Scattergram of postnatal age plotted in relationship to the reduction of RR by 0.1 or 1.0 μM R-PIA and 50 μM dipyridamole.(B) Relationship between postnatal age and the inhibition of RR by R-PIA 0.1 (<48 h, n = 6, ≥48 h, n = 5) or 1.0 μM(n = 6 and 7, respectively) or 50 μM dipyridamole (n = 5 and 8, respectively). The effects of the adenosine agonist and the nucleoside transport inhibitor dipyridamole were correlated to postnatal age, with a more pronounced effect in the younger animals. (C) Theophylline, 55 μM (n = 5 and 9, respectively), increased the RR compared with control and could reverse the depression of respiratory activity if applied together with 1 μM R-PIA (R-PIA +Theophylline) (n = 5 and 5, respectively). Error bars are SEM (*p < 0.05).

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Because it was only in preparations from younger (<48 h) pups that adenosinergic drugs could elicit an irregular breathing pattern and because the RR and Ti did not differ significantly (except in two(0-2 h) preparations), we divided the pups into two age groups. For analysis we thus compared the data both altogether and divided into two groups,i.e. preparations from pups with a postnatal age of 1) less than 48 h (<48 h, n = 27) and 2) of more than 48 h(≥48 h, n = 35) (Table 2).

Table 2 Drugs and age groups: experimental design
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Adenosine receptor agonist decreases RR. A representative recording of the respiratory activity after administration of R-PIA is shown in Figure 2B. In all preparations, the respiratory activity was depressed during administration of 1 μM R-PIA (n = 13), mainly due to a reduction of the RR (-23 ± 4%, p < 0.01). Neither Ti (-14 ± 12%) nor the mean C4/C5 burst amplitude (-5 ± 6%) was significantly altered, although in 2 of 13 individual preparations a significant decrease in amplitude was observed. At a 10-fold lower concentration, i.e. 0.1 μM (n = 11), R-PIA also decreased the RR (-12.9 ± 0.7%, p < 0.05). The Ti (3 ± 12%) and the C4/C5 burst amplitude (-0.8± 2%) did not change significantly (Fig. 4B).

Figure 4
figure 4

Irregular respiration induced by an adenosine agonist. In preparations from rats younger than 48 h, the effects on the RR were pronounced, and a reversible irregular RR could be induced. (A) The respiratory activity recorded with a preparation from a 20-h-old rat in standard solution. (B) As in (A), after 22 min of perfusion with 0.1 μM R-PIA. (C) As in (B), after 22 min of perfusion with 1 μM R-PIA. (D) As in (C), after 22 min of recovery with standard solution. C4, respiratory activity.

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A block of adenosine uptake decreases C4/C5 respiratory activity. Upon application of the adenosine uptake blocker dipyridamole (50μM; n = 13), the respiratory activity decreased in a manner similar to that seen with R-PIA (RR -26 ± 4.2%, p < 0.01;Ti -10 ± 6.5%) as shown in Figure 3B. The C4/C5 burst amplitude did not change significantly when the data were pooled, but decreased significantly in three and increased significantly in one preparation.

Endogenous adenosine decreases C4/C5 respiratory activity. Theophylline (55 μM; n = 14) increased the RR (13 ± 2.1%) compared with controls (p < 0.01), whereas no significant change was observed in Ti (0.4 ± 3.8%). A significant increase in the C4/C5 burst amplitude was observed in two of the preparations, but in most cases (12/14) no significant increase was seen. DPCPX (50 μM;n = 5) had a similar effect, RR increased; 13.4 ± 1.8%,p < 0.01 and Ti did not change; 1.5 ± 8.4. None of the preparations had significant changes in the Int. C4/C5 amplitude. No significant difference between the effects of theophylline and DPCPX application was found (unpaired t test).

The effects of R-PIA and that of dipyridamole on fi, RR, CV, Ti, and amplitude could all be reversed or prevented by administration of theophylline or DPCPX. When 1 μM R-PIA was applied together with 55 μM theophylline (n = 10), no significant changes in RR (-2.8 ± 2.1%), CV (-3.6 ± 12.6%), amplitude (+4.7 ± 3.9%), or Ti (-7.2 ± 7.6%) were observed compared with the control (Figs. 2D and 5B).

An adenosine receptor agonist and uptake blocker decrease activity of I neurons (fi). Application of 1 μM R-PIA significantly decreased fi in 9 out of 10 I neurons examined (Table 3). When dipyridamole was applied, the fi decreased significantly in all three of the I neurons examined; 17.7 ± 1.6 spikes/s during control conditions and 6.9± 1 spikes/s (p < 0.01) during dipyridamole perfusion.

Table 3 Effect of R-PIA and theophylline on the intraburst frequency (fi) of I neurons
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Theophylline increased the fi in all four of the I neurons examined, although this effect was statistically significant only in two individual experiments. For the total data this value was 16.1 ± 1.0 spikes/s under control conditions to 18.4 ± 1.5 spikes/s during theophylline perfusion. The effects of R-PIA on fi could be reversed by administration of theophylline together with R-PIA(Table 3).

DPCPX increased the fi in all three of the I neurons examined. The control value was in this case 14.5 ± 1.7 spikes/s compared with 18.7 ± 3.4 spikes/s during exposure to DPCPX.

No differences, at any given age, in control, drug, or post-drug C4/C5 respiratory parameters were detected between preparations in which I neuronal activity was recorded and preparations in which only C4/C5 activity was recorded. We therefore consider the I neuronal recordings to be representative for the whole population of animals.

Age-dependent effects of adenosine receptor agonist and uptake blocker on respiratory activity. As shown in Figure 5, R-PIA had a significantly more pronounced effect on RR in preparations from younger pups (<48 h) compared with those from older pups (≥48 h). This age-dependent difference in the effect on RR was significant with both 0.1 and 1.0 μM R-PIA (RR change; Spearman rank correlation; p = 0.003,n = 11 and p = 0.025, n = 12, respectively). The change in fi caused by 1.0 μM R-PIA also tended to be age-dependent, although this trend was not statistically significant (age versus fi change; Spearman rank correlation; p = 0.1 n = 10). When the two age groups <48 h and ≥48 h were compared, no significant age dependency was seen with regard to fi change (Table 3). Neither Ti nor the C4/C5 burst amplitude demonstrated any significant dependency on age. Like R-PIA, dipyridamole had a more pronounced effect on preparations from the youngest rats (RR change; age versus drug Spearman rank correlation; p = 0.02, n = 13)(Fig. 5). The effects of theophylline and DPCPX on RR,Ti, and fi showed no dependency on age.

Age-dependent effects of adenosine receptor agonist and antagonists on the regularity of respiratory rhythm. The effect of R-PIA on the change of CV was found to depend significantly on age when all preparations were compared (CV control/CV drug; Spearman rank correlation;p = 0.004, n = 24). When comparing the age groups <48 h(0.55 ± 0.24, n = 12) with ≥48 h (1.21 ± 0.24,n = 12) no significant difference could be shown (p = 0.06, unpaired t test). Most (9/12) of the preparations from the younger pups (2-48 h) responded with a significantly more irregular breathing pattern when R-PIA was applied. In 6 of these 12 preparations, a respiratory pattern with clustered respiratory bursts, similar to the respiratory pattern of the youngest pups (CV 0.50 + 0.07), appeared (Fig. 4, A-C). In preparations from pups older than 48 h, R-PIA induced a significant increase in CV in 3/12 preparations compared with control, but in total there was no significant effect on CV. In this age group a respiratory pattern with clustered respiratory bursts could be evoked in only one preparation, and even in this case, a higher (10 μM) concentration of R-PIA was required to obtain this effect. When comparing the number of experiments in which R-PIA could induce a increase in CV, a significant difference between the two age groups (<48 h/≥48 h) was found (two-tail Fischer's exact test, p = 0.04).

With DPCPX the same tendency for age dependency was observed; <48 h (CV control/CV DPCPX 2.72 ± 1.27 n = 3) ≥48 h (CV control/CV DPCPX 0.67 ± 0.31 n = 2). However, the number of DPCPX experiments were not sufficient to make conclusions regarding DPCPX-induced changes of CV and age dependency. The effect of theophylline on the change of CV was, as with that of R-PIA, found to depend significantly on age when all preparations were compared (CV control/CV theophylline; Spearman rank correlation; p = 0.04, n = 13). When comparing the age groups <48 h (CV control/CV theophylline; 1.32 ± 0.18, n = 5) and ≥48 h (CV control/CV theophylline; 0.76 ± 0.15, n = 8) theophylline increased the regularity of RR compared with control in the young group significantly more than in the older age group (t test independent means, p = 0.04, n = 13).

DISCUSSION

We have found that the adenosine A1 receptor agonist R-PIA and the adenosine uptake blocker dipyridamole depress respiratory neuronal activity in the ventrolateral structures of the rostral medulla, as well as decreasing the frequency of respiratory bursts in brainstem-spinal cord preparations from the neonatal rat. This depression was reversed by theophylline and the specific A1 receptor antagonist DPCPX. Furthermore, we found that the reduction in RR by R-PIA and dipyridamole is inversely correlated to postnatal age.

Adenosine has a direct central depressive effect on RR. It has previously been suggested that adenosine decreases breathing by lowering body temperature and thus reducing the oxygen demand(22). Instead, our results corroborate with earlier findings in in vivo models of adult cat and rat, suggesting that both adenosine and xanthine derivatives have direct effects on the respiratory neural network in the brainstem(32, 33). Furthermore, our results indicate that adenosine decreases inspiratory neuronal activity and respiratory output by the activation of A1 receptors in the neonatal rat. This is in accordance with findings in adult cats demonstrating modulation of expiratory neurons and synaptic transmission by adenosine(34). Our present in vitro findings clearly demonstrate that adenosine inhibits respiration, not only via an indirect effect on body temperature and metabolism, but also by direct action on the functional network of respiratory neurons within the medulla oblongata.

Endogenous adenosine depresses respiration. The finding that the nucleoside uptake blocker, dipyridamole, inhibited respiratory activity in a manner similar to that of the A1 receptor agonist R-PIA suggests that endogenous adenosine has a modulatory effect on central respiratory control mechanisms.

This is further supported by the consistent increase in respiratory output by the receptor antagonists theophylline and DPCPX. Taken together, these findings imply that in this in vitro preparation, as is the case in vivo(34), respiration is modulated by the endogenous activation of adenosine receptors under control conditions.

Adenosine A1 receptors mediate the adenosinergic respiratory depression. The findings of responses to the unspecific theophylline and dipyridamole similar to the responses to specific A1 antagonist/agonists support the conclusion that the A1 receptor contributes significantly to the observed modulation of respiratory activity. However, we cannot exclude the possibility that adenosine A2 receptors may be involved in the observed responses to dipyridamole or theophylline.

Adenosine A1 receptors reduce presynaptic Ca2+ influx(35), which subsequently inhibits the release of excitatory transmitters(36), such as glutamate(37). Furthermore, postsynaptic A1 receptors can hyperpolarize the neuron by activating potassium channels(38). Such pre- and postsynaptic inhibition of neuronal activity could explain the observed depression of respiratory activity. One might speculate that this pre- and postsynaptic inhibition of neuronal activity and an ensuing decrease in activation of the voltage-dependent NMDA channel could explain the irregular breathing induced by the adenosine agonist in vitro. This is in agreement with the finding that blocking the glutaminergic NMDA receptor can induce irregular or apneustic breathing patterns in vivo(39, 40).

Earlier studies have shown that presynaptic A1 receptors modulate synaptic transmission to phrenic motoneurons(23). Furthermore, theophylline can attenuate both the hypoxic depression of respiration in vivo(5, 19) and the depression of C4 motoneuronal discharge in vitro(41). Adenosine could be a link between cellular energy metabolism(42) and the excitability of neurons, because breakdown of ATP increases substantially during hypoxia and more adenosine is formed. Therefore, the actions of adenosine on respiration-related neurons in the brainstem shown here, together with the observations on spinal motoneurons might explain, part of the respiratory decrease induced by hypoxia(43, 44).

Theophylline in high concentrations can increase cAMP levels by inhibiting phosphodiesterase and the excitatory effects of theophylline on respiration might thus be explained by such a rise in cAMP concentration. However, higher concentrations of theophylline than the ones used in the present study are required to affect phosphodiesterase in vitro(45). Furthermore, it has previously been reported that forskolin, which increases cAMP levels, has no effect on the RR in the brainstem-spinal cord preparation when the control RR is >5 bursts/min(46). We therefore suggest that the responses to theophylline observed in our study, can be attributed primarily to a blockade of other A1 receptor-mediated effects, rather than to a decrease in cAMP.

Age and the effect of adenosine on respiration. The effects of both R-PIA and dipyridamole on RR were found to be significantly different between the two postnatal age groups, i.e. 0-48 h and ≥48 h (Fig. 5). Furthermore, both the agonist of the adenosine A1 receptor and the blocker of adenosine uptake could induce reversible irregular breathing patterns in preparations from the yonger rat pups (Fig. 4).

The irregularity of RR increases with adenosine receptor agonists and uptake blocker in inverse relation to postnatal age. Furthermore, the irregularity of RR decreases with antagonists in inverse relation to postnatal age. These data indicates that adenosine is one of the factors contributing to the irregular respiratory pattern observed.

The instability of breathing in the newborn period has been attributed to the development of neuronal networks(47). It seems likely that such an immature neuronal network could be more susceptible to the influences of an inhibitory modulator such as adenosine.

This correlation between postnatal age and drug effects has not been described previously with this in vitro preparation. We therefore stress the importance of taking age into account when interpreting data from this neonatal preparation. Our results are in agreement with earlier findings in neonatal rabbits(5) and piglets(48), where a considerably more pronounced effect of adenosine agonists on respiration was found in newborn (1-3 d) compared with older animals (8 d and 3 wk). Thus, an increased sensitivity to adenosinergic agents in the neonatal rat immediately after birth could explain our observations.

The effects of theophylline on respiratory activities were not, however, found to be age-dependent, indicating that adenosinergic modulation under control conditions is the same in the newborn as in the 2-4-d-old pups. We are not able to explain this finding at present.

Irregular breathing can be induced in human infants by hypoxia(12) and reversed by theophylline, suggesting that adenosine released as a result of the hypoxia is involved. In the present study the effects of both the adenosine agonist R-PIA and dipyridamole could be reversed by theophylline, which is used in neonatal practice to treat apnea and irregular breathing. The present data supports the suggestion that the therapeutic effect of theophylline on recurrent neonatal apnea is due to its antagonism of central adenosine receptors(49).

Medullary respiratory neurons are responsible for respiratory central pattern generation(50, 51). Central application of an adenosine agonist or an uptake blocker to the in vitro preparation, where no afferent feedback is preserved, causes depression of both medullary respiratory neuronal units and the RR.

Thus, our data suggest that, in addition to its role at the level of the spinal cord(23), the adenosinergic system is also active within the brainstem and is involved in the modulation of respiratory neurons and the RR. We thus suggest that the changes in 1) inspiratory neuronal activity, 2) C4/C5 respiratory burst rate, and 3) irregularity caused by adenosinergic agents observed here are mediated at the level of the medulla oblongata.

In conclusion, we have found that adenosine depresses the activity of inspiratory-related neurons in the neonatal rat medulla oblongata as well as decreasing the RR and the regularity of respiratory rhythm. The potency of this modulatory effect of adenosine on respiration decreases during the first days after birth. Furthermore, theophylline can block this respiratory depression, primarily by acting on adenosine A1 receptors in the medulla.