2C receptor activation are reflected in the developmental pattern of fos expression in the brainstem

Brain Research 942 (2002) 51–57 www.elsevier.com / locate / bres

Research report

Postnatal changes in the respiratory response of the conscious rat to serotonin 2A / 2C receptor activation are reflected in the developmental pattern of fos expression in the brainstem Florence Cayetanot*, Franc¸oise Gros, Nicole Larnicol ´ , 3 rue des Louvels, 80036 Amiens Cedex 01, France Laboratoire de Neurophysiologie, ETPAPC, EA 2088, UFR de Medecine Accepted 21 February 2002

Abstract The influence on the breathing pattern of the activation of serotonin receptors belonging to the subtypes 2A and 2 C (5-HT 2A / 2C ) has been assessed in newborn and adult conscious rats. Rats were given an acute intraperitoneal dose of the agonist DOI (1-(2.5-dimethoxy-4iodophenyl)-2-aminopropane; 5 mg / kg). In newborns, DOI elicited a long-lasting respiratory depression by decreasing both tidal volume and respiratory frequency. In adults, DOI retained a depressant influence, although attenuated, on tidal volume. In contrast, it elicited an increase in respiratory frequency. In separate subsets of newborn and adult rats, immunohistochemistry has been used to monitor c-fos expression induced by DOI in the medullary and pontine regions involved in respiratory control. Counts of immunoreactive neurons indicated a marked increase in the neuronal populations activated in the adult compared to the newborn rat. The response to both experimental factors (newborn vs. adult controls) and drug (injected vs. control age-matched rats) were more pronounced in mature animals. Among developmental changes in the pattern of labeling, DOI elicited Fos expression in the adult but not in the neonate in the ¨ ventrolateral subnucleus of the nucleus of the solitary tract, the parabrachial area and the Kolliker-Fuse nucleus. This finding suggested that changes in the respiratory response to DOI might at least partly depend on maturational events within networks involved in the modulation of respiratory timing.  2002 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Respiratory regulation Keywords: Serotonin; 5-HT 2 receptor; Respiration; Fos expression; Postnatal development; Rat

1. Introduction Serotonin has been involved in many aspects of ventilatory control. Of the different families of serotonin receptors, 5-HT 2 receptors have been the subject of thorough investigations on their influence on the pattern of breathing. Based on both in vitro and in vivo preparations, those studies suggested that serotonin binding to 5-HT 2 receptors might influence ventilation either positively or negatively, depending on the target neurons and the components of the respiratory control networks present in the preparation (see

*Corresponding author. Tel.: 133-322-82-7698; fax: 133-322-827947. E-mail addresses: [email protected] (F. Cayetanot), [email protected] (N. Larnicol).

[4,9] for reviews). For instance, it has been demonstrated in neonatal brainstem–spinal cord preparations that the activation of 5-HT 2A / 2C receptors led to a facilitation of spinal motoneurons related to respiratory muscles, but that it depressed hypoglossal inspiratory activity [17]. However, this latter finding has not been corroborated by recordings from medullary rhythmic slices [1]. Data in anaesthetized adult rats suggested that the apnea elicited by 5-HT was partly mediated by the activation of 5-HT 2 receptors distributed to the vagal afferent pathway and / or to the airways [26]. On the other hand, L-tryptophan loads, which increased endogenous 5-HT levels and consequently favored serotonin binding to both peripheral and central 5-HT 2 receptors, elicited apnea in the newborn but not in the adult anesthetized rat [10]. Hence, both studies in the anaesthetized animal suggested that 5-HT 2 receptor mechanisms might be involved in the pathogenesis of obstruc-

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02690-2

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tive apnea but pointed out that this response might depend on the maturational status. Although the recurrence of apneas, chiefly during sleep, is related to significant morbidity in infants and adult humans, the involvement of 5-HT 2 -receptor mechanisms in respiratory disturbances occurring under physiological conditions has not yet been documented. Hence, the primary objective of the present study was to describe the respiratory response to the acute systemic administration of DOI (1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane), a 5-HT 2A / 2C agonist, in newborn and adult free-moving rats. This analysis was extended by the description of the pattern of Fos expression evoked by DOI within regions of the medulla and the pons related to cardiorespiratory control. Fos, the protein product of the immediate early gene c-fos, can be detected by immunohistochemistry and is commonly used as a trans-synaptic metabolic marker of neurons activated within polysynaptic pathways triggered by a variety of stimuli in the conscious, intact animal [16]. Notably, DOI caused a localized Fos expression in forebrain regions previously demonstrated to contain 5-HT 2 binding sites [15]. Moreover, we recently showed that the developmental pattern of Fos expression could provide useful indications about the functional status of neuronal relays at different postnatal ages [3]. Thus, Fos immunohistochemistry appeared as a valuable approach to the neuronal basis of the respiratory response to 5-HT 2A / 2C receptor activation and of its postnatal changes. Part of the data has already been published in abstract form [5].

2. Materials and methods The experimental protocols were carried out in conformity with the European Communities Council Directive (86 / 609 / EEC) on 28 newborn (0–3 days) and 20 adult Sprague–Dawley rats of either sex (220–305 g). DOI (1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane; RBI) was given at a single dose of 5 mg / kg dissolved in normal saline. This dose was selected on the basis of existing published information [15] and of pilot series. The drug solution was prepared immediately before use and injected intraperitoneally as a single bolus (0.05 ml in newborns, 0.1 ml in adults). DOI was deliberately given intraperitoneally rather than subcutaneously to minimize the risk that the interpretation of maturational changes in the response to 5-HT 2A / 2C activation might be biased by changes in the rate of absorption and / or distribution of the agonist [8]. Control animals were injected intraperitoneally with saline. In both newborn and adult rats, ventilatory variables and Fos expression were studied in separate subsets of five to eight animals. Control and DOI-injected rats used for recording ventilatory variables were transferred to an experimental

chamber in which they could freely move. The chamber was connected to a differential transducer (Validyne DP45, sensitivity: 62 cmH 2 O) to record the pressure changes induced by the respiratory flow, using a modification of the plethysmographic technique described by Bartlett and Tenney [2]. The chamber was continuously flushed with humidified air between the recording periods and maintained at temperatures close to the thermoneutral zone (32 8C in newborns, 21–25 8C in adults). Frequency and tidal volume were evaluated at fixed intervals of 5 min, on a minimum of 10 respiratory cycles, and for a maximum of 1 h in the neonates and 2 h in the adults, to avoid any stress-related effect of starvation or dehydration. Signals were digitized through a SPIKE2 (CED) data analysis system and stored on disks. The measurements performed during the 15 min preceding either saline or DOI injections were used as indices of baseline respiratory frequency and tidal volume. Further changes in ventilatory parameters were expressed as percentages of baseline value, fixed at 100%. At the end of the experiments, the rats were either sacrificed or were returned to their cage for checking their ability to recover from treatment. Animals used for Fos immunohistochemistry were maintained in their home cage. They were sacrificed 2 h after injection by an overdose of pentobarbital (100 mg / kg) containing heparin (250 I.U. / kg). They were fixed in situ by transcardial perfusion of 0.1% xylocain in saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brainstems were removed, postfixed overnight, and stored in cryoprotectant until freeze sectioning. Fos immunohistochemistry was conducted on free-floating coronal sections of the brainstem (40 mm thick) as routinely done in the laboratory [3]. Briefly, every second (in newborns) or third (in adults) section was incubated for 48 h in a rabbit polyclonal antibody raised against Fos synthetic peptide (Santacruz, diluted 1 / 4000). Fos-like immunoreactivity was detected by the avidin–biotin technique using commercial kits (Vectastain Elite, Vector Labs.) containing a biotinylated goat anti-rabbit IgG and an avidin–biotinylated peroxidase complex (ABC). Peroxidase activity was revealed by 0.02% diaminobenzidine (DAB) in the presence of 0.01% H 2 O 2 and enhanced by 0.04% nickel ammonium sulfate. This procedure led to light background staining, sufficient for not requiring further counterstaining. In every animal, all of immunostained sections were examined under light microscopy. Anatomical localization and regional nomenclature were defined according to Paxinos and Watson [19]. The distribution of Fos-like immunoreactive (FLI) neurons was plotted onto drawings performed with the aid of a drawing tube attached to the microscope (objective 34 or 310). Labeled nuclei were counted at higher magnification (objective 320) using an eyepiece fitted with a squared grid to avoid neurons to be counted more than once. Counts were performed in

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individual anatomical structures along their entire longitudinal extent. The sizes of neuronal populations per anatomical structure were estimated at 23 (in newborns) or 33 (in adults) the total sum of neurons counted, as previously described [3]. For each structure, group averages of (i) the numbers of sections containing FLI neurons, (ii) the numbers of FLI neurons per section, and (iii) the estimates of total population sizes were calculated together with the standard errors of the means (S.E.M.). Data on respiratory and Fos responses to DOI administration were analyzed by Wilcoxon signed-ranks test on individual values in each group ( STATVIEW1 package). They were considered significant at P,0.05. For Fos data, both mean numbers of immunoreactive neurons per section and total population sizes were considered.

3. Results

3.1. Effect of DOI on respiratory variables 3.1.1. Newborns In newborn rats, baseline respiratory frequency was 133615 breaths / min. Controls (n56) tended to respond to saline injection by a transient increase in respiratory frequency, which did no reach the level of significance. In contrast, DOI injections (n56) elicited a long-lasting reduction in ventilation. Respiratory frequency and tidal volume reached their minimal value after 20 (67% of baseline) and 35 min (55% of baseline), respectively, and remained decreased until the end of the recording period (Fig. 1). 3.1.2. Adults The average breathing rate of adult rats was 98.463.0 breaths / min before injection. Neither respiratory frequency nor tidal volume was significantly affected by saline injection in the control group. The adult respiratory response to DOI markedly differed from that in newborns (Fig. 2). DOI elicited an immediate increase in frequency which peaked at 165% of predrug value 15 min after injection. Thereafter, frequency gradually recovered baseline values. As in newborn rats, DOI elicited a steady decrease in tidal volume. Nevertheless, this decrease was smaller in adults than in newborns, as the amplitude of the pressure signal never fell below 80% of baseline value. 3.2. Fos-like immunoreactivity evoked by DOI administration Compared to saline, DOI increased Fos expression in several medullary and pontine areas devoted to respiratory and autonomic control. Differences between neonates and adults indicated marked developmental changes in the pattern of labeling (Table 1) (Fig. 3).

Fig. 1. Changes in respiratory frequency (A) and tidal volume (B) following systemic administration of DOI (black columns; n58) or saline (open columns; n58) to the newborn rat, at time 0. Data are expressed as mean percents of the baseline (6S.E.M.), averaged from successive intervals. *, Significantly different from control value.

3.2.1. Newborns After DOI injection, the mean number of Fos-like immunoreactive (FLI) neurons per section was significantly above control values in the commissural subdivision of the nucleus of the solitary tract (NTSc), the rostroventrolateral medulla (RVL) and the lateral paragigantocellular nucleus (LPGi). The analysis of population sizes also demonstrated a significant effect of DOI on Fos expression in the NTSc and LPGi, but not in the RVL. 3.2.2. Adults Neurons expressing Fos in response to DOI were more widely distributed in adult than in newborn rats. The number of FLI neurons was above control values in both the commissural and ventrolateral subnuclei of the nucleus of the solitary tract (NTSc, NTSvl). In the ventral medulla, DOI induced Fos expression in the caudoventrolateral medulla (CVL). Fos expression was not significantly enhanced in the LPGi, which contrasted with the newborn. In the pons, the number of FLI neurons was significantly above control values in the locus coeruleus (LC), in the

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Fig. 2. Changes in respiratory frequency (A) and tidal volume (B) following systemic administration of DOI (black columns; n55) or saline (open columns; n55) to the adult rat, at time 0. Data are expressed as mean percents of the baseline (6S.E.M.), averaged from successive intervals. *, Significantly different from control value.

lateral (LPB) and medial (MPB) subdivisions of the ¨ parabrachial area, as well as in the region of the KollikerFuse nucleus (KF). The estimation of population sizes indicated that the number of FLI neurons was higher in adults than in neonates in most areas studied, and in both saline- and DOI-injected rats. These developmental changes varied in magnitude with the regions under study. They were especially marked in the LPGi and in the parabrachial area where a seven- to tenfold increase in the number of FLI neurons was observed between 0 and 3 days and maturity.

4. Discussion The present study demonstrated that the respiratory response to the activation of 5-HT 2A / 2C receptors by systemic injection of DOI undergoes marked developmental changes. DOI acted as a respiratory depressant in the neonate, while it elicited tachypnea in adult rats. These changes might reside in maturational events that were

mirrored in increased neuronal Fos expression in the medulla and the pons. Data in newborns corroborated our recent findings that the intraperitoneal injection of DOI led to hypoventilation in conscious newborn rats, by a concomitant reduction of both breathing rate and tidal volume [5]. A comparable decrease in breathing frequency has been observed in response to the application of DOI to the floor of the 4th ventricle in decerebrate newborn rats [13]. As DOI crosses the blood–brain barrier, it seems conceivable that the response of the intact neonate might at least partly be related to the activation of central 5-HT 2A / 2C receptors. In further support of this hypothesis, Onimaru et al. [18] demonstrated that both applications of 5-HT 2C receptor agonists to in vitro neonatal brainstem preparations slowed down the respiratory-like rhythmic activity recorded from cervical roots. Using the same preparation, Morin et al. [17] demonstrated that DOI depressed hypoglossal inspiratory activity. Thus, central 5-HT 2 receptor mechanisms might also be accounted for by the reduction in tidal volume we observed in the intact neonate. Nevertheless, the contribution of peripheral mechanisms should also be considered, as they have been involved in the decrease in phrenic activity and lung compliance observed during apnea induced by 5-HT in the adult anesthetized rat [26]. To our knowledge, we were the first to document the respiratory response to the intraperitoneal injection of DOI in the conscious adult rat. Under the conditions we used, DOI elicited an abrupt increase in breathing frequency, later associated with a decrease in tidal volume. Thus, the activation of 5-HT 2A / 2C receptors apparently retains the same influence on tidal volume throughout postnatal development, while it elicits opposite changes in the rate of breathing in newborn and adult rats. It should be noted that the increase in respiratory frequency presently described contrasts with the apnea evoked by the intravenous administration of a-methyl-5-HT, another 5-HT 2 agonist, to the anaesthetized rat [26]. This discrepancy suggests that the balance between excitatory and inhibitory influences of 5-HT 2 receptor activation might be altered by anesthesia. Furthermore, this balance is likely to depend on intrinsic characteristics of the agonists tested, such as the fraction that crosses the blood–brain barrier and their greater selectivity for a specific 5-HT 2 receptor subtype. The estimation of the size of FLI neuron populations indicated that the developmental change in the respiratory response to 5-HT 2A / 2C receptor activation was associated with an increase in neuronal responsiveness not only to this specific stimulation but also to side events such as handling and / or injection. Indeed, the difference between DOI-injected rats and their age-matched controls was consistently larger in adults than in neonates, even in the LPGi where it lost its significance in adults. In salineinjected controls, FLI neurons were also significantly more numerous in adults than in neonates in several structures. Notably, control Fos expression underwent a tenfold

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Table 1 Fos expression in the medulla and the pons of newborn and adult rats Newborn

Adult

Control n58

DOI n58

Control n55

DOI n55

nTSc

Count / sect n Population

75.7619.7 4.060.6 618.86197.1

140.8627.8* 6.260.5 1432.56413.6*

54.7610.4 9.261.4 1388.46207.2

119.1610.7* 10.460.9 3718.26473.0* [

nTSvl

Count / sect n Population

41.269.7 3.660.3 304.0682.0

65.3610.4 3.660.5 535.06201.7

22.368.5 5.060.3 309.06104.0

54.166.1* 5.860.9 931.26136.8* [

LRt

Count / sect n Population

38.765.1 5.861.2 522.56122.1

37.765.6 8.060.5 418.56262.0

24.162.5 10.261.3 734.46115.5

62.7614.4* 12.060.6 2157.06391.7* [

CVL

Count / sect n Population

29.766.4 3.760.4 207.3649.3

47.5612.4 3.660.5 396.56114.0

13.461.0 8.861.3 357.06145.6 [

24.363.4* 10.461.0 760.26309.4*

RVL

Count / sect n Population

19.665.5 5.661.2 290.06100.1

36.968.2* 4.861.2 397.46142.6

14.863.6 9.860.9 438.46196.1

30.766.0 9.260.8 859.26196.1 [

LPGi

Count / sect n Population

4.161.5 3.660.8 68.5614.2

15.462.8* 4.861.1 126.0645.6*

9.462.2 17.661.4 506.46146.0 [

12.362.9 18.261.7 718.26211.3 [

LC

Count / sect n Population

18.263,9 3.460.7 80.3612.8

21.063.7 3.860.9 84.3615.8

29.0610.5 10.061.7 637.86170.8 [

53.5610.1* 8.061.0 1213.2625.7* [

LPB

Count / sect n Population

16.262.7 2.761.0 98.0634.0

12.963.2 3.060.8 89.1644.0

38.764.4 6.860.7 756.6633.6 [

87.4617.9* 6.860.6 1749.86386.0* [

MPB

Count / sect n Population

4.561.1 1.761.0 12.869.2

5.060.2 2.160.7 17.5610.3

8.060.9 6.860.7 160.8620.7 [

28.264.1* 6.860.6 583.56108.9* [

KF

Count / sect n Population

14.761.9 6.260.4 279.0646.9

41.164.0* 6.060.4 734.3673.4*

Not identifiable in the neonate

Values are given as group averages6S.E.M.; count / sect, number of FLI neurons / 40 mm section; n, number of sections examined which contained FLI neurons throughout individual structures; population, estimate of the total number of FLI neurons in individual structures (see Methods). *, [: indicate significant differences between stimulated and age-matched control rats, and between newborn and adult rats, respectively. nTSc, nTSvl, commissural and ventrolateral subnu. of the solitary tract; LRt, lateral reticular nucleus; CVL, RVL, caudal, ventrolateral medulla; LPGi, lateral ¨ paragigantocellular nu; LC, locus coeruleus; LPB, MPB, lateral, medial parabrachial area; KF, Kolliker-Fuse nu.

increase in the parabrachial area from birth to maturity, corroborating our earlier findings in nonhandled rats [3]. However, control Fos expression was not influenced by the age in the NTS vl, the LRt, the CVL and the RVL. Hence, postnatal changes in the respiratory response to 5-HT 2A / 2C receptor are likely the result of complex interactions between the developmental regulation of 5-HT 2 receptor levels and the overall maturation of functional connections within central networks, including those devoted to breathing control. On the basis of evaluations on whole brain membrane preparations (Fig. 5 in [22]), 5-HT 2A receptor levels are about twice as high in adults as in neonates, while 5-HT 2C levels keep rather steady throughout postnatal development. Furthermore, it is generally agreed that the neural networks controlling respiration are not fully established at birth. Among recent evidence, it has been

demonstrated that most of the synaptic development occurs postnatally within key medullary relays of breathing control pathways (NTS, nucleus ambiguous and VLM) [21], and that postnatal alterations in the morphology of NTS neurons parallel changes in passive properties and spike characteristics [24]. A noticeable feature of the pattern of labeling was that DOI enhanced Fos expression in the NTS vl and in distinct subdivisions of the parabrachial area in the adult but not in the neonate. Both regions have been reported to contribute to respiratory phase switching in the adult rat [6,14,25]. This suggests that developmental changes in the respiratory response to DOI might reveal maturational events within networks regulating respiratory timing. Nevertheless, the exact nature of these events remains to be elucidated. Indeed, although Fos expression may argue

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periphery, notably to the airways. Thus, the possibility that the developmental changes we observed might partly reside on peripheral mechanisms cannot be ruled out. (iii) Finally, the duration of respiratory phases, and consequently breathing rate, is highly regulated not only by ascending inputs originating from the periphery (e.g. vagal receptors) [4] but also by descending inputs from suprapontine origin [11]. The development of these central influences might also contribute to postnatal changes in the respiratory response to DOI administration, in the way that they have been involved in the maturation of the response to hypoxia [3,12,23].

Acknowledgements This work was supported by the University of Picardie and the French Ministry of Research and Technology.

References

Fig. 3. Line drawings of individual sections from an adult (A) and a newborn rat (B) following systemic administration of DOI, showing the ¨ distribution of Fos-positive neurons to the parabrachial area, the KollikerFuse nucleus and the locus coeruleus. Abbreviations: LPB, lateral ¨ parabrachial area; MPB, medial parabrachial area; KF, Kolliker-Fuse nu.; LC, locus coeruleus; scp, brachium conjunctivum. Scale bar50.5 mm.

in favor of a relationship between the activation of solitary and parabrachial neurons and the disappearance of the depressant influence of DOI on breathing frequency, these changes raised some issues. (i) Assuming that they are the primary consequence of an increased level of 5-HT 2A / 2C receptor expression, it should be remembered that Fos expression could result from trans-synaptic stimulation. Hence, it cannot be ascertained whether the neurons identified in the present study might bear 5-HT 2A / 2C receptors or whether they might represent relays within multisynaptic pathways triggered by DOI, via distant central and / or peripheral receptors. The earlier finding that the density of 5-HT 2 receptor-binding sites was low in the pons and the medulla would support the latter possibility [20]. However, the presence of diffusely distributed presynaptic 5-HT 2A receptors has recently been described in ¨ the parabrachial area, the Kolliker-Fuse nucleus and, at a lesser extent, in the NTS of adult rats [7]. (ii) Another point is that central 5-HT 2A / 2C receptors appeared to be functional in the neonatal rat, even if fewer than in adults [22]. In contrast, there are no data available on the ontogeny of 5-HT 2A / 2C receptors distributed to the

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