Proceedings of The Physiological Society

University of Cambridge (2004) J Physiol 555P, PC13

Communications

The HCN1 ion channel subunit is prominently expressed in somatodendritic domains of neurones in sensory and motor systems

R.E. Brooke, I.J. Edwards, C.J. Milligan, S.A. Deuchars and J. Deuchars

School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK


Hyperpolarization-activated cyclic nucleotide-gated (HCN) nonselective cation channels in neurones carry cationic currents proposed to generate Ih (Robinson & Siegelbaum, 2003). Several CNS regions express the mRNA for one or more of the 4 HCN subunits. Here we show that immunoreactivity for HCN1 is present in neurones which underlie sensory or motor functions.

Rats (100-200 g, n = 10) were injected intraperitoneally with 0.1ml 1 % Fluorogold (Fluorochrome Inc.) and 7 days later were humanely killed by intraperitoneal injection of Sagatal (60 mg kg-1) and transcardial perfusion with 4 % paraformaldehyde/0.1-0.5 % glutaraldehyde. Slices (50µm) of brainstem, spinal cord, dorsal root ganglia (DRG) and nodose ganglia (NG) were cut on a vibrating microtome and incubated in rabbit anti-HCN1 antibody (Alomone;1:1K) followed by Cy3 conjugated donkey anti-rabbit (Jackson Immunochemicals, 1:500). For double labelling sections were subsequently immersed in a marker for unmyelinated c-fibres, fluorescein conjugated Bandeiraea (Griffonia) Simplicifolia Lectin I (IB4, VectorLabs, 1:100) and/or an indicator of fast conducting myelinated neurones, mouse anti-neurofilament 200 (NF200, 1:1K, Sigma) visualised using a biotinylated conjugated secondary antibody and Streptavidin Alexa488.

HCN1-immunoreactivity (HCN1-IR) was strongly evident throughout the brainstem and spinal cord, including the following motor nuclei: facial, abducens, ambiguus, hypoglossal and ventral horn. These HCN1-IR neurones were also Fluorogold and/or NF200 containing, confirming that they were motor neurones. HCN1-IR was also in neurones of sensory pathways, namely in the cochlear, spinal trigeminal, dorsal column, inferior olivary and lateral spinal nuclei, as well as the area postrema and the dorsal horn. On a cellular level HCN1-IR was present in somatic and dendritic membranes. Primary sensory neurones that were HCN1-IR and Fluorogold containing were detected in the mesencephalic trigeminal nucleus, the NG and DRG. These primary sensory neurones were IB4 negative, but a proportion did contain NF200.

These data show that the HCN subunit HCN1 is present in somato-dendritic regions of neurones which subserve sensory and motor functions. This localisation parallels the expression of Ih in neurones within these populations (e.g. McLarnon, 1995), suggesting that the HCN1 subunit is a contributor to channels mediating these currents.

This work was funded by the British Heart Foundation (REB, SAD), Wellcome Trust (CJM) and a Physiological Society vacation studentship (IJE).

Where applicable, experiments conform with Society ethical requirements