Proceedings of The Physiological Society

University College Cork (2004) J Physiol 560P, PC3



Milligan,Carol J; Edwards,Ian J; Brooke ,Ruth E; Deuchars,Jim ;

1. School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) non-selective cation channels in neurones carry cationic currents proposed to perform diverse functions, such as, generation of rhythmic activity and may underlie the hyperpolarisation activated Ih current. (Pape, 1996). The 4 HCN subunits are differentially expressed in the CNS. Here we examined the distribution of HCN1 channel subunits in the rat brainstem, using immunohistochemistry. Male rats (150-250g) were humanely killed by intraperitoneal injection of Sagatal (60mg/kg i.p.) followed by transcardial perfusion with 0-4% paraformaldehyde in 0.1M phosphate buffer. Autonomic preganglionic and motor neurones were pre-labelled by intraperitoneal injection of 0.1ml of 0.1% of Fluorogold (Fluorochrome Int.) or application of tracers to the superior laryngeal nerve 3-7 days prior to perfusion. Brainstem sections (50μm) were incubated in either rabbit anti-HCN1 (1:1000 Alomone) with goat anti-choline acetyltransferase (ChAT, 1:500, Chemicon) or mouse anti-non-phosphorylated neurofilament 200 (NF200, clone N52, Sigma, UK) antisera. All motor neurone pools in the medulla oblongata contained HCN1-immunoreactivity (IR). In the nucleus ambiguus (NA) HCN1-IR neurones contained Fluorogold and ChAT-IR indicating that they have peripherally projecting axons and synthesize acetylcholine, but not all ChAT or FG cells contained HCN1-IR. HCN1-IR in the NA was present only in NF200-IR cells, suggesting that it is expressed in motor but not autonomic preganglionic neurones. The localisation of these HCN1-IR cells was also consistent with motor neurones – they were present in the semi-compact formation of the NA that contains neurones innervating the striated muscle of the larynx and pharynx. Curiously, the compact formation of the NA was unlabelled with HCN1-IR even though it also contains motor neurones, whose target is the oesophagus. However, these cells were NF200 negative, suggesting that they are not α motor neurones. Preliminary experiments indicate that HCN1-IR is present in neurones specifically labelled retrogradely from the superior laryngeal nerve. HCN1-IR may therefore be a useful immunohistochemical marker to specifically identify motor neurones in the NA. Since cells projecting to the larynx display patterns of activity in vivo related to the respiratory cycle it will be of interest to determine if these motor neurones are the prime source of respiratory neurones with Ih recorded in the pre-Botzinger complex (Mironov et al. 2000).

Where applicable, experiments conform with Society ethical requirements