Birth-Related Activation of Preprotachykinin-A mRNA in the Respiratory Neural Structures of the Rabbit

003 1-3998191/2904-0369$03.00/0 PEDIATRIC RESEARCH Copyright 0 I991 International Ped~atricResearch Foundation, Inc. Vol. 29. No. 4, 199 1 Printc.d in liS A. Birth-Related Activation of Preprotachykinin-A mRNA in the Respiratory Neural Structures of the Rabbit Tlrc Nol1c4 Institutc,fi~rNc~~~rophv.siolo~y[M.S., Y.Y., H.L.] AND Department ~ f M e d i c a lClrc.mi.stry /II.P./, Luhorufor,~ c!fMoleculur ~Veurohialogy.Karolinslca Institutet and Department ofPdiatric.s, Karolinslca Ilospifnl S-104 01, S~oekholrn,S~vc>den ABSTRACT. The perinatal ontogeny of preprotachykininA gene expression was assessed in central respirationrelated structures. On the day of birth, there was an enhanced expression of preprotachykinin-A mRNA in the region of the nucleus tractus solitarii, the primary relay station for respiratory and cardiovascular reflexes. This increased expression was also seen in the pups delivered by cesarean section and allowed to breathe for a couple of hours as compared with their littermates, which were not allowed to breathe at all. On the basis of this finding, we suggest that the commencement of continuous breathing at birth, unlike the episodic breathing of fetal life, is associated with the enhanced expression of preprotachykinin-A mRNA in the nucleus tractus solitarii. (Pediatr Res 29: 369-371,1991) Abbreviations DRG, dorsal respiratory group nTS, nucleus tractus solitarii PPT-A, preprotachykinin-A STR, striatum E31, 1 d before expected delivery PO, day of birth P I . . . P8, number of days after birth Birth is a momentous event for the fetus. The ability to cope in the new environment requires the establishment of effective continuous respiration. Although the fetus demonstrates breathing movements, these are episodic in nature (1). Respiration is partially inhibited at this stage and poorly controlled by metabolic needs. This inhibition has been postulated to be due to a dominance of inhibitory neuromodulators in fetal life (2, 3). At birth, there is an increase of excitatory neuroactive agents (4). Among the latter is the tachykinin, substance P, which is known to stimulate respiration (5). We have earlier shown that substance P causes a more pronounced increase in ventilation in the youngest animals as compared with older ones (6). Substance P, first isolated by von Euler and Gaddum (7), belongs to a family of structurally related peptides termed the tachykinins. It is abundantly present both in the central and peripheral nervous systems (8). The other mammalian tachykiReceived May 3. 1990; accepted October 30. 1990. Correspondence: M. Srinivasan, The Nobel Institute for Neurophysiology, Karolinska Institutet, S-104 O I Stockholm, Sweden. Supported in part by research grants from National Institute for Lung and Heart Diseases SRC (29) I ROl HL 39921-01, Expressen's Prenatal Foundation, Swedish National Science Research Council. Swedish Medical Research Council. No. 19X5234, and the Heart and Lung Foundation. nins are neurokinin-A and neurokinin-B. Substance P and neurokinin-A are encoded by the same gene, PPT-A. PPT-A gene undergoes alternative splicing to give rise to three forms, of which a PPT-A mRNA encodes only for substance P, whereas the Pand 7-PPT-A mRNA encode for both substance P and neurokinin-A (9, 10). A recent study demonstrated the presence of PPT-A mRNA-containing cells in the nTS and none in the cerebellum (1 1). Significant quantities of substance P-like immunoreactivity was found to be localized in the nTS (12), which plays an important role in the reflex transmission of visceral afferent inputs. The region of the nTS has been shown to receive multiple inputs from peripheral sources such as respiratory, cardiovascular, gustatory, and gastrointestinal systems via the V, VII, IX, and X cranial nerves (13, 14). Substance P, which is abundant in nTS, is known to mediate baro- and chemoreceptor inputs to the nTS; hypoxic provocation results in an increased release of substance P in the region of nTS (15). Denervation of the IX and X cranial nerves decreases the immunoreactivity for substance P in the nTS (16). Thus, substance P has been suggested to have a significant physiologic role in chemo- and baroreceptor transduction mechanisms. Two distinct populations of substance P-immunoreactive neurons have been described in the ventrolateral medulla: I) a rostra1 group situated ventral to the facial nucleus and 2) a caudal group lying ventrolaterally spanning the rostro-caudal length of the inferior olivary nucleus (17). In a developmental study on substance P immunoreactivity in the rabbit nTS using the technique of RIA, higher levels have been shown in younger animals than in adults (18). However, the time points just around birth are lacking in this study. Therefore, a study on the ontogenetic expression of PPT-A mRNA was undertaken especially around the period of birth in rabbit pups. Rabbits, which are known to be precocial developers, closely resemble human infants with regard to the maturation of sleep-wakefulness cycles (19). MATERIALS AND METHODS Animals. Rabbit pups of various ages (E31, PO, P1, P3, P8, and adult) were killed with an overdose of pentobarbital. The following regions, depicted in Figure 1, were rapidly dissected out, frozen on dry ice, and stored at -70°C until processed further: 1) the DRG containing the region of the nTS (however, it should be noted that this area does not specifically contain only nTS and such a resolution could have been obtained by combining with in situ hybridization) where the chemoreceptor afferents terminate; 2) the ventral medullary surface structures where the central chemoreceptors are thought to be located; 3) STR as a hybridizing control; and 4) cerebellum as a negative control area. In the second set of experiments, pregnant rabbits were anes- 370 SRlNIVASAN ET AL thetized and pups were delivered by cesarean section a day before estimated delivery. Half the pups in the litter were immediately killed, whereas the rest of the pups were placed in a warm environment and allowed to breathe for 2 h. Prior permission from the Animal Ethical Committee, Stockholm, was obtained for carrying out these experiments. RNA preparatzon and blot analyszs. The dissected regions from four to six pups were pooled for each age. Frozen tissue samples were homogenized with a Polytron in 4 M guanidine isothiocyanate, 0.1 M 8-mercaptoethanol, 0.025 M sodium citrate, pH 7.0. Each homogenate was layered over a 4-mL cushion of 5.7 M CsCl in 0.025M sodium citrate, pH 5.5, and centrifuged at 15°Cin a Beckman SW41 rotor at 35 000 rpm for 2 1 h (20). The recovery of RNA was quantified spectrophotometrically before use in RNA blot analysis. RNA from each sample (30-50 pg) was electrophoresed in 1% agarose gels containing 0.7% formaldehyde and transferred to nitrocellulose filters as previously described (21). The filters were prehybridized in 4 x SSC (1 x SSC = 0.15M NaC1, 0.0 15M sodium citrate pH 7.0) 40% formami (...truncated)