Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC231
Immunohistochemical localisation of the ??1 Na+/K+-ATPase subunit in the mouse spinal cord and brainstem
L. Howe1, C. Lawrenson1, J. Deuchars1
1. University of Leeds, Leeds, United Kingdom.
The Na+/K+-ATPase (NKA) enzyme is a ubiquitous membrane protein complex contributing to the resting membrane potential in excitable cells. NKA is a heterodimer of both α and β subunits. In the CNS, α1 and α3 are the predominant α-subunit isoforms, yet despite their potentially different kinetic contributions to the enzyme, there is a paucity of knowledge as to their anatomical distribution and localisation to different cell types. Currently, there is conflicting evidence on the distribution of α1NKA in the spinal cord (McGrail et al, 1991; Watts et al, 1991). Here the α1NKA was localised using fluorescence immunohistochemistry. Adult C57BL/6 mice (n=5) were injected i.p. with the retrograde tracer Fluoro-gold (FG; Fluka, BioChemika, USA) 24 h prior to being anaesthetised with sodium pentobarbital (60mg/kg; i.p.) and perfused transcardially with 4% paraformaldehyde (Milligan et al, 2006). Brainstem and spinal cord were sectioned at 50µm on a vibrating microtome. α1NKA was localised using an antibody raised against α1 subunit (Epitomics Inc, Burlingame, USA). At all levels of the spinal cord, α1NKA-immunoreactivity (α1NKA-IR) was observed in the membrane of neuronal somata and dendrites in the ventral horn. These were α-motoneurones as they were FG and NF200+ve. Sympathetic preganglionic neurones (SPNs) in the interomediolateral cell column were also α1NKA-IR. Co-localisation of α1NKA and synaptic vesicular protein (SV2) seen in the dorsal horn at all levels of the spinal cord indicates that α1NKA is also present pre-synaptically. Intense α1NKA-IR was also observed in the central canal ependymal cells. In the brainstem, α1NKA-IR was concentrated in motoneurones in the hypoglossal nucleus, compact and semi-compact nucleus ambiguus, spinal trigeminal nucleus, trigeminal motor nucleus, facial nucleus as well as the Raphé nuclei (n=5). A small percentage of cells (10-20%) in the mesencephalic nucleus of the trigeminal nerve (Me5) were α1NKA-IR (n=3), their location consistent with that of periodontal mechanoreceptors (Lazarov, 2007). In the abducens nucleus (nVI), trochlear nucleus (nIV) and oculomotor nucleus (nIII), α1NKA-IR was observed in a small percentage of neurones which were parvalbumin-negative, indicating that they project to multiply innervated muscle fibres in the extraocular muscles (Eberhorn et al, 2005). This study reveals expression of α1NKA in a-motoneurones, indicating a role in setting the resting membrane potential of excitable cells which control motor function of the trunk and limbs as well as head, neck and face. In addition, in the spinal cord α1NKA-IR was also detected in SPNs and presumptive sensory afferent terminals in the dorsal horn.
Where applicable, experiments conform with Society ethical requirements