Maturational changes in pontine and medullary alpha-adrenoceptor influences on respiratory rhythm generation in neonatal rats

Abstract

We examined developmental changes in α-adrenoceptor influences and descending pontine inputs on the medullary respiratory network in the neonatal rat in vitro brainstem-spinal cord preparation. Using a split bath preparation to isolate the pons from the medulla, antagonists for α1 and α2 adrenoreceptors were applied to only the medulla at postnatal days 0, 2 and 4, before and after transection of the pons. Blocking α1 and α2 receptors in the medulla in the absence of a pons reduced burst frequency at all ages with a more pronounced effect in younger animals. At all ages the presence of a pons diminished the effect of blocking α2 receptors in the medulla and eliminated the effect of blocking α1 receptors. These results indicate that there is a tonic release of catecholamines within the medulla that is under influence from the pons. Additionally, transection experiments indicated that during development, the net influence of the pons changed from one of excitation to one of inhibition.

Introduction

Extensive studies using isolated medullary preparations support the existence of a respiratory rhythm generator (RRG) in the rostral ventrolateral medulla (Suzue, 1984, Smith et al., 1991, Onimaru and Homma, 2003). Additionally, it has been shown that there are neurons with respiratory-related activity in the rostral pons (see Bianchi et al., 1995, Alheid et al., 2004 for reviews) and while the pons may not be necessary for rhythm generation, it is important in modulating and shaping respiratory motor output. One known method of pontine modulation of respiratory motor output is via release of catecholamines.

The medullary RRG receives both tonic noradrenergic inhibitory and excitatory drives originating, respectively, from the A5 and A6 noradrenergic nuclei of the pons (Hilaire et al., 1989, Errchidi et al., 1990, Errchidi et al., 1991). It has been postulated that A5 neurons project to and release noradrenaline (NA) onto inhibitory α2-adrenoreceptors in the medulla while A6 neurons project to and release NA onto excitatory α1-adrenoreceptors (Hilaire et al., 2004). There is evidence that both types of neurons are present at birth and are required for proper development of the respiratory rhythm generators, although the net balance of the two receptor types varies across species and developmental age (Shirasawa et al., 2000, Viemari et al., 2004, Viemari et al., 2005a, Hilaire et al., 2004). More recent evidence suggests there is also a medullary source of catecholamines that modulates breathing in addition to the traditionally described pontine sources (Zanella et al., 2006).

In the literature, there is a general agreement that the pons provides a net inhibitory input onto the medulla in early neonates. There is dispute, however, as to whether this inhibition decreases during development (Errchidi et al., 1991), or whether it persists through adulthood (Jodkowski et al., 1997, Dawid Milner et al., 2001). Further arguments have been made not only for the attenuation of pontine inhibitory influences during development, but also for an increase in pontine excitatory drive to the medullary RRG. Morphological studies showed that the number of A5 neurons decreased as age increased (between 1 and 10 days post-natal) (Ito et al., 2002) and it has been suggested that this contributes to the decrease in pontine inhibition. Although Ito and colleagues found no change in the volume of the A6 area, Hakuno et al. (2004) showed that excitatory effects of A6 neurons on the respiratory rhythm increased with age and integrating this with the previous findings from Ito et al. (2002) and Errchidi et al. (1991), suggested there was a shift during early development in the relative roles of the “inhibitory” A5 area and the “excitatory” A6 area.

In contrast to the results described above, where pontine inhibition appeared to decrease with age in rats, Fong et al. (2008) suggested that pontine inhibition increases with age as removal of the pons in in vitro brainstem-spinal cord (en bloc) preparations progressively increased frequency in neonatal rat pups from day of birth (P0) until 4 days post partum (P4). These findings parallel observations made in mouse preparations (Viemari et al., 2003) where pontine inhibition was mild at embryonic days 16–18 (E16–E18) and became especially potent at E18–P5 where it abolished breathing.

Given the discrepancies in reports of developmental changes in pontine inputs to the medullary RRG in neonatal rodents, and the suggestion that not all catecholaminergic input to the rhythm generator originates in the pons (Zanella et al., 2006), the present series of experiments was designed (1) to revisit this issue using narrowly defined age groups, (2) to clarify the mechanisms by which the effects of pontine, and specifically catecholaminergic, input change during early development and (3) to determine whether there are developmental changes associated with catecholaminergic inputs to the rhythm generator coming from the medulla itself. To achieve this we used two variations of the neonatal en bloc preparation: the pontomedullary preparation which contained the pontine A5 and A6 groups in addition to the medullary A1/C1 and A2/C2 groups, and the medullary preparation which retained only the medullary catecholaminergic groups.