Disease-specific human glycine receptor alpha1 subunit causes hyperekplexia phenotype and impaired glycine- and GABA(A)-receptor transmission in transgenic mice

Hereditary hyperekplexia is caused by disinhibition of motoneurons resulting from mutations in the ionotropic receptor for the inhibitory neurotransmitter glycine (GlyR). To study the pathomechanisms involved in vivo, we generated and analyzed transgenic mice expressing the hyperekplexia-specific dominant mutant human GlyR alpha1 subunit 271Q. Tg271Q transgenic mice, in contrast to transgenic animals expressing a wild-type human alpha1 subunit (tg271R), display a dramatic phenotype similar to spontaneous and engineered mouse mutations expressing reduced levels of GlyR. Electrophysiological analysis in the ventral horn of the spinal cord of tg271Q mice revealed a diminished GlyR transmission. Intriguingly, an even larger reduction was found for GABA(A)-receptor-mediated inhibitory transmission, indicating that the expression of this disease gene not only affects the glycinergic system but also leads to a drastic downregulation of the entire postsynaptic inhibition. Therefore, the transgenic mice generated here provide a new animal model of systemic receptor interaction to study inherited and acquired neuromotor deficiencies at different functional levels and to develop novel therapeutic concepts for these diseases

Spinal inhibitory synaptic transmission in the glycine receptor mouse mutant spastic

Inhibitory glycine receptor (GlyR) and GABA(A) receptor (GABA(A)R)-mediated synaptic transmission was examined in two strains of the GlyR mutant mouse spastic and the respective wild types. The mutants display a mild and a severe neurological phenotype. Electrically evoked postsynaptic whole-cell currents were recorded from alpha-motoneurons in lumbar spinal cord slices. Amplitudes of GlyR-mediated IPSCs were significantly reduced in the severe phenotype in comparison to the respective wild type and the mild phenotype mutants. Surprisingly, amplitudes of GABA(A)R-mediated IPSCs were also significantly reduced in both mutants. Fast time constants of the decay phase of IPSCs were slightly reduced for the GlyR-mediated IPSCs and significantly larger for the GABA(A)R-mediated IPSCs in both mutant strains

Altered potassium channel function in the superficial dorsal horn of the spastic mouse

The spastic mouse has a naturally occurring glycine receptor (GlyR) mutation that disrupts synaptic input in both motor and sensory pathways. Here we use the spastic mouse to examine how this altered inhibitory drive affects neuronal intrinsic membrane properties and signal processing in the superficial dorsal horn (SDH), where GlyRs contribute to pain processing mechanisms. We first used in vitro patch clamp recording in spinal cord slices (L3–L5 segments) to examine intrinsic membrane properties of SDH neurones in spastic and age-matched wildtype controls (∼P23). Apart from a modest reduction (∼3 mV) in resting membrane potential (RMP), neurones in spastic mice have membrane and action potential (AP) properties identical to wildtype controls. There was, however, a substantial reorganization of AP discharge properties in neurones from spastic mice, with a significant increase (14%) in the proportion of delayed firing neurones. This was accompanied by a change in the voltage sensitivity of rapid A-currents, a possible mechanism for increased delayed firing. To assess the functional consequences of these changes, we made in vivo patch-clamp recordings from SDH neurones in urethane anaesthetized (2.2 g kg−1, i.p.) spastic and wildtype mice (∼P37), and examined responses to innocuous and noxious mechanical stimulation of the hindpaw. Overall, responses recorded in wildtype and spastic mice were similar; however, in spastic mice a small population of spontaneously active neurones (∼10%) exhibited elevated spontaneous discharge frequency and post-pinch discharge rates. Together, these results are consistent with the altered intrinsic membrane properties of SDH neurones observed in vitro having functional consequences for pain processing mechanisms in the spastic mouse in vivo. We propose that alterations in potassium channel function in the spastic mouse compensate, in part, for reduced glycinergic inhibition and thus maintain normal signal processing in the SDH

Probing glycine receptor stoichiometry in superficial dorsal horn neurones using the spasmodic mouse

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