Postnatal changes in the noradrenergic system modulating hypoglossal motoneurons

Abstract

Appropriate activation of hypoglossal (XII) motoneurons (MNs) during inspiration is essential for maintaining upper airway patency. Morphological and electrophysiological properties of XII MNs, as well as numerous modulatory systems affecting these MNs, undergo significant change postnatally. These changes must be properly coordinated to ensure that breathing continues uninterupted throughout development. We have previously described an α1 noradrenergic receptor-mediated potentiation of XII nerve activity that increases approximately 1.8-fold over the first three postnatal days and 2.5-fold over the first two postnatal weeks [1]. To examine the development of the noradrenergic system underlying this potentiation, we combined electrophysiological and immunohistochemical approaches. Mechanisms underlying the potentiating effects of α1 receptor activation were assessed using whole-cell recordings from XII MNs in rhythmically-active medullary slice preparations from P0 (postnatal day zero) and P3 mice. The development of the catecholaminergic cell phenotype and the time course over which norepinephrine (NE) immunoreactive terminals appear in the XII nucleus were examined immunohistochemically using a monoclonal antibody against tyrosine hydroxylase (TH; Boehringer Mannhiem) and a polyclonal antibody against NE (Chemicon) in mice ranging from P0 to adult. Tissue from all age groups was processed simultaneously and labelled using a stan-protocol. Images were captured using a CCD dard DAB/H2O2 camera and optical density of immunolabelling was measured using NIH Image software. Phenylephrine (PE) induced an inward current or a depolarization in all inspiratory XII MNs tested (n = 21). At a holding potential of -60 mV, currents induced by PE (100 μM) increased from 2.9 ± 0.7 pA/pF in P0 (n = 9) to 4.7 ± 1.1 pA/pF in P3 (n = 12)MNs. Application of PE did not affect input resistance (RN) in P0 MNs (n = 7) but increased RN by 11 ± 4% in P3 MNs (n = 11). PE potentiated inspiratory synaptic currents, and the magnitude of this potentiation was significantly greater in P3 (peak current 25 ± 7%; charge transfer per burst 43 ± 7%, n = 8) relative to P0 (peak 17 ± 7; charge transfer per burst 28 ± 11%, n = 6) XII MNs. Involvement of post-synaptic receptors was indicated by persistence of the responses in TTX (1 μM, n = 7). PE did not affect repetitive firing elicited by injected current in P0 MNs tested (n = 2). However, the frequency/current relationship was significantly shifted to the left in 3/5 P3 MNs. Immunohistochemical data showed that labelling for TH in the A5, A7 and LsC (Locus subCoeruleus) cell groups was already strong in P0 and increased approximately 30% by P14, before decreasing to adult levels which were similar to P0 values. Immunolabelling for NE in these cell groups was also apparent at P0. A developmental increase in NE immunolabelling was modest, except in LsC where it increased up to 140% by P7, before decreasing to adult levels (that were half those observed in P0 animals). The number of NE-positive fibers and varicosities within XII nucelus was low in P0 mice, but increased dramatically by P14 where dense innervation was present along the entire rostro-caudal extent of the nucleus. In adults, NE-positive terminals/varicosities were dense over only a limited rostro-caudal region. These data suggest that the factors contributing to the developmental increase in the noradrenergic potentiation of XII MN activity include a proliferation of NE-containing terminals within the XII nucleus and maturation of postsynaptic α1 receptor mechanisms.