Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse
Article first published online: 13 MAY 2011
© 2011 The Authors. Journal compilation © 2011 The Physiological Society
The Journal of Physiology
Volume 589, Issue 10, pages 2459–2474, May 2011
How to Cite
Graham, B. A., Tadros, M. A., Schofield, P. R. and Callister, R. J. (2011), Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse. The Journal of Physiology, 589: 2459–2474. doi: 10.1113/jphysiol.2011.206326
- Issue published online: 13 MAY 2011
- Article first published online: 13 MAY 2011
- (Received 27 January 2011; accepted after revision 4 March 2011; first published online 8 March 2011)
Non-technical summary Glycine receptors (GlyRs) play an important role in inhibiting neurone activity in the spinal cord. Until recently adult GlyRs were thought to comprise α1 and β subunits. A new form of the receptor containing α3 subunits has been discovered in the superficial dorsal horn (SDH), a region of the spinal cord important for pain. This raises questions about the precise subunit composition of GlyRs and glycinergic synapses in the SDH. We used the spasmodic mouse, where α1 subunit containing GlyRs have altered agonist sensitivity and electrophysiological properties, to ask how α1 and α3 subunits are assembled to form GlyRs on SDH neurones. We found most (∼75%) GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design pain drugs that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.
Abstract Inhibitory glycine receptors (GlyRs) are pentameric ligand gated ion channels composed of α and β subunits assembled in a 2:3 stoichiometry. The α1/β heteromer is considered the dominant GlyR isoform at ‘native’ adult synapses in the spinal cord and brainstem. However, the α3 GlyR subunit is concentrated in the superficial dorsal horn (SDH: laminae I–II), a spinal cord region important for processing nociceptive signals from skin, muscle and viscera. Here we use the spasmodic mouse, which has a naturally occurring mutation (A52S) in the α1 subunit of the GlyR, to examine the effect of the mutation on inhibitory synaptic transmission and homeostatic plasticity, and to probe for the presence of various GlyR subunits in the SDH. We used whole cell recording (at 22–24°C) in lumbar spinal cord slices obtained from ketamine-anaesthetized (100 mg kg−1, i.p.) spasmodic and wild-type mice (mean age P27 and P29, respectively, both sexes). The amplitude and decay time constants of GlyR mediated mIPSCs in spasmodic mice were reduced by 25% and 50%, respectively (42.0 ± 3.6 pA vs. 31.0 ± 1.8 pA, P < 0.05 and 7.4 ± 0.5 ms vs. 5.0 ± 0.4 ms, P < 0.05; means ± SEM, n= 34 and 31, respectively). Examination of mIPSC amplitude versus rise time and decay time relationships showed these differences were not due to electrotonic effects. Analysis of GABAAergic mIPSCs and A-type potassium currents revealed altered GlyR mediated neurotransmission was not accompanied by the synaptic or intrinsic homeostatic plasticity previously demonstrated in another GlyR mutant, spastic. Application of glycine to excised outside-out patches from SDH neurones showed glycine sensitivity was reduced more than twofold in spasmodic GlyRs (EC50= 130 ± 20 μm vs. 64 ± 11 μm, respectively; n= 8 and 15, respectively). Differential agonist sensitivity and mIPSC decay times were subsequently used to probe for the presence of α1-containing GlyRs in SDH neurones. Glycine sensitivity, based on the response to 1–3 μm glycine, was reduced in >75% of neurones tested and decay times were faster in the spasmodic sample. Together, our data suggest most GlyRs and glycinergic synapses in the SDH contain α1 subunits and few are composed exclusively of α3 subunits. Therefore, future efforts to design therapies that target the α3 subunit must consider the potential interaction between α1 and α3 subunits in the GlyR.