Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC232
Immunohistochemical localisation of the ??3 Na+/K+-ATPase in the brainstem and spinal cord: clues to pathogenesis in rapid-onset dystonia-parkinsonism.
C. Lawrenson1, L. Howe1, S. Clapcote1, S. A. Deuchars1, J. Deuchars1
1. The University of Leeds, Leeds, United Kingdom.
Rapid-onset dystonia parkinsonism (RODP) is associated with heterozygous missense mutations in the alpha 3 subunit of Na+/K+-ATPase (NKAα3) (Carvalho et al, 2004). To determine a possible locus for the mutations in NKAα3 to influence sensorimotor processing the NKAα3 was localised in spinal cord, brainstem and sensory ganglia of mice. Adult C57BL/6 mice (n=8) were anaesthetised with sodium pentobarbital (60mg/kg; i.p.) and perfused with 4% paraformaldehyde. 3-5days prior to perfusion mice were injected i.p with Fluoro-gold (0.1ml of 1%, FG; Fluka, BioChemika, USA) to label motor, preganglionic and sensory neurones. Brainstem, spinal cord and dorsal root ganglia (DRG) sections were cut for immunohistochemistry to 50µm using a vibrating microtome. In the spinal cord NKAα3 (1:1000, Upstate Biotechnology) was localised to γ-motoneurones, identified as Choline Acetyltransferase (ChAT; 1:500, Chemicon) and FG positive but NeuN (1:1000, Millipore) negative (Friese et al, 2009). NKAα3 immunoreactivity (IR) co-localised with Kv2.1 (1:50, NeuroMab) but not synaptic vesicle protein-2 (SV2; 1:1000), indicating postsynaptic localization on γ-motoneurones. In contrast α-motoneurones were NKAα3 immunonegative, although apposing SV2-IR terminals contained NKAα3-IR. The facial nucleus, which does not contain γ-motoneurones (Welt and Abbs, 1990), was NKAα3 immunonegative (n=3). All spinal cord ventral horn Renshaw cells (n=3, 39/39 cells) identified with calbindin (1:1000, SWANT), also contained NKAα3-IR. In DRG the number of FG positive cells expressing NKAα3 from DRG at C4 level (mean 73.14±13.18%) was significantly higher (P<0.05 n=3) than in L2 (mean 17.17±6.0%). These NKAα3-IR cells were proprioceptive cells as they contained Neurofilament-200 (1:1000, Sigma; 24±7% NF200-IR were NKAα3-IR, n= 3 mice; n=27 sectiones) or parvalbumin (1:1000, Sigma; n=3; 26±17% of PV cells were NKAα3-IR, n=3 mice, n= 19 sections). In the brainstem the mesencephalic trigeminal nucleus (Me5) contains proprioceptive cells (Lazaroz, 2007), which were also NKAα3-IR (n=5). The muscle spindle peripheral endings in mouse masseter muscles were also NKAα3-IR (n=3). These results indicate that in sensorimotor reflex pathways the NKAα3 is expressed in proprioceptors and γ-motoneurones. Mutations in the NKAα3 which lead to RODP decrease NKAα3 function, suggesting that increased excitability of the proprioceptive and γ-motoneurones may underlie the dystonic like symptoms of patients with such mutations.
Where applicable, experiments conform with Society ethical requirements