Amyotrophic lateral sclerosis (ALS) is accompanied by muscle fasciculation and spasticity. Changes in membrane excitability of motor neurones may contribute to the disease progression of ALS (Bostock et al. 1995) and, given a possible role in precipitating neuronal degeneration in ALS, represent a target for therapeutic intervention. We have therefore investigated the ‘excitability state’ of motor neurones maintained in culture.

Using whole-cell and cell-attached patch-clamp methods we investigated spontaneous electrical activity in primary spinal cord cultures, isolated from humanely killed Balb-C mice (E13). The bath contained Locke solution (mM: 140 NaCl; 3 KCl; 2 MgCl₂; 2 CaCl₂; 10 Hepes; 10 glucose; buffered to pH 7.4 using NaOH). The recording pipette contained (mM): 140 KCl; 5 NaCl; 1 MgCl₂; 1 CaCl₂; 11 EGTA; Hepes 10; buffered to pH 7.2 with KOH. Successful giga-ohm seals were formed in 68/116 cells (seal resistance 1-4 GV).

Whole-cell measurements indicated that motor neurones in culture have healthy resting potentials (RMPs; mean ± S.D. -57 ± 11 mV; n = 19). In the cell-attached configuration, spontaneous biphasic waveforms, known as action currents (ACs), were observed at 0 mV pipette potential (RMP) in 40 % of cells (n = 148). We have recorded these ACs over pipette potentials -60 to +40 mV. Often inward single channel potassium currents followed the ACs (n = 7). The presence of ACs at RMP was not age dependent and interestingly activity was of two types. Cells fired continuously and/or in bursts. The frequency of firing was voltage dependent and the interspike interval clearly decreased with patch hyperpolarisation (n = 8). Furthermore, the intraburst interval decreased with patch hyperpolarisation (n = 6). The action currents were completely abolished in the presence of 1 µM TTX. Glutamate receptor antagonists (100 µM NBQX and D-AP5) reduced the overall number of cells firing to 28 % (n = 20).

The resting membrane potential values and spontaneous electrical activity we have observed here indicate that the neurones are fully functional within our culture system (Latham et al. 2000). Spontaneous action currents, are indicative of freely changing membrane potential within the neurones. Our results suggest that a percentage of these cells may be ‘pacemaker’ cells which continue to fire without glutamatergic input. Such features of cultured motor neurones may provide reliable indices against which to test pharmacological intervention.