Hedgehog pathway were all significantly hyperpolarized by 2 3 mV in the 2 M riluzole condition. Hyperpolarization of the RMP would indicate hypoexcitability, while hyperpolarization of the PIC and NaPIC onsets and max voltage would lead to hyperexcitability. Unexpectedly, the PIC amplitude was significantly decreased 25% in the 2 M condition, and there was a nonsignificant 13% decrease in the NaPIC amplitude. Therefore, chronic exposure to riluzole did not induce a compensatory upregulation of the PIC or NaPIC, contrary to our predictions. Although not the main aim of this study, extrinsic excitability did not appear to increase in the riluzole treated cells. In the absence of synaptic blockers, spontaneous firing was not increased and there was no evidence for the development of bursting behavior. Synaptic scaling, however, was not directly tested and remains a possibility. Interactions between genotype and drug treatment. Although acute riluzole application has similar PLK effects on wt and SOD1 neurons, prolonged riluzole treatment produced differential effects. In addition to the main effect of drug treatment on PIC amplitude, there was a significant interaction between genotype and drug treatment.
Post hoc analysis revealed that the PIC amplitude between pimobendan control and riluzole treated SOD1 neurons was significantly decreased by 55.4%, but there was no change between control and riluzole treated wt neurons. Likewise, the NaPIC amplitude between control and riluzole treated SOD1 neurons was decreased 23%, although this was not significant. The decrease in PIC amplitude did not, however, lead to decreases in FI gain or changes in other firing parameters. Reapplication of riluzole. Although chronic riluzole treatment did not have major effects on motoneuron output, it was important to test whether riluzole could still decrease motoneuron excitability or if neurons had become desensitized to it. For 11 neurons chronically treated with riluzole, 2 M riluzole was added to the aCSF after initial measurements were taken. Subsequently, TTX was added to the external solution, allowing the comparison between the NaPIC amplitude before and after reapplication of 2 M riluzole. For two neurons, riluzole was washed out of the ofloxacin aCSF before TTX was added. The reapplication of 2 M riluzole either decreased or stopped AP firing during current ramps for both wt and SOD1neurons.
During 1 s current steps, the neurons fired repetitive APs but at a slower rate and AP failure occurred before the end of the step. However, there was little to no change in the initial AP height during the current step. The NaPIC amplitude was decreased an average of 61.5% when 2 M riluzole was reapplied, which is similar to the acute effects of 2 M riluzole in other studies. Therefore neurons did not become desensitized to riluzole after prolonged exposure. It is unlikely that the initial reduction in PIC amplitude for chronically drug treated SOD1 neurons was due to residual effects of riluzole in the aCSF. First, in aCSF without synaptic blockers, there was no significant difference between spontaneous firing rates for drug treated and untreated neurons. Second, in two of two neurons, riluzole was quickly washed out of the bath after reapplication and repetitive firing returned to normal.