Proceedings of The Physiological Society
University of Leeds (2002) J Physiol 544P, S003
The potassium channel subunit Kv3.1b is expressed in both GABAergic and glutamatergic neurones in the nucleus tractus solitarii (NTS) of the adult rat
C.J. Milligan and J. Deuchars
School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK
Kv3.1b is a member of the Shaw family of potassium channels with rapid activation and deactivation kinetics, commonly expressed in fast-firing GABAergic neurones (Erisir et al. 1999). We have previously reported the presence of the Kv3.1b subunit in a subset of neurones in the NTS of the adult rat medulla oblongata (Deuchars & Atkinson, 2001). Here we examine the possibility that these Kv3.1b-expressing neurones are GABAergic.
Male rats (100-150 g) were anaesthetised with Sagatal (60 mg kg-1 I.P.), perfused transcardially with artificial cerebrospinal fluid in which NaCl was replaced with sucrose (217 mM) and the brain removed. Total RNA was isolated from medulla and reverse transcribed, followed by 35 cycles of PCR performed using specific oligonucleotide primers for glutamic acid decarboxylase (GAD65/67), vesicular glutamate transporter 2 (VGLUT2) or Kv3.1. For in situ hybridisation rats were anaesthetised as above and perfused transcardially with 4 % paraformaldehyde in 0.1 M phosphate buffer. Coronal 30 mm sections of medulla were hybridised with digoxigenin-UTP-labelled GAD65, GAD67 or VGLUT2 sense and antisense RNA probes using a modified version of the manufacturer's protocol (www.biochem.roche.com). Subsequently, sections were incubated in a polyclonal antibody that recognises the carboxy terminal (residues 567-585) of the Kv3.1b subunit (Alomone). Sections were then washed and placed in either donkey anti-rabbit conjugated Cy3 (1:1000, Jackson) or a biotinylated secondary anti-rabbit IgG (1:200, Vector), followed by streptavidin Alexa (1:1000, Molecular Probes). Sections were then prepared for light microscopic analysis.
RT-PCR reveals the presence of GAD65, GAD67, VGLUT2 and Kv3.1b transcripts in the rat medulla. Furthermore, in situ hybridisation shows mRNA for GAD65 and GAD67 in GABAergic neurones and mRNA for VGLUT2 in glutamatergic neurones throughout the medulla. Kv3.1b immunoreactivity (IR) was also observed throughout the medulla. In the NTS, Kv3.1b IR was present in neurones in the ventrolateral, interstitial, medial and dorsolateral subnuclei where the staining appeared to be membrane bound. A small proportion of NTS neurones positively labelled for GAD65, GAD67 or VGLUT2 mRNAs were also immunoreactive for the Kv3.1b subunit. These results clearly demonstrate that the Kv3.1b potassium channel subunit is expressed in both GABAergic and glutamatergic neurones in the NTS of the adult rat medulla oblongata, which are likely to have fast firing phenotypes.
Thanks to Ruth Stornetta for the VGLUT2 clone and A. Tobin for the GAD65 and GAD67 clones. This work was supported by The Wellcome Trust.
All procedures accord with current UK legislation.
Where applicable, experiments conform with Society ethical requirements