In order to investigate the potential role of endogenously produced NO in the modulation of the intrinsic properties of spinal motor neurons, the nitronyl nitroxide NO scavenger, PTIO, was bath-applied to the lumbar spinal cord slices while recording from presumed motor neurons.
PTIO is a NO scavenger, referred to as membrane permeable as it is considerably more lipophilic than the carboxy analogue (cPTIO) and therefore will scavenge NO in both extra- and intra-cellular environments. Recordings from ventral roots showed that removal of endogenous NO during fictive locomotion by PTIO caused an increase in locomotor frequency, possibly by inhibition of excitatory output from the CPG, and an increase in locomotor amplitude, by scavenging NO and preventing activation of sGC at the level of the last order interneurons or directly at the motor neuron (Chapter 3, Section 3.3.2). These experiments suggest that NO has an endogenous modulatory role in the locomotor network; thus, PTIO was used to investigate changes to the intrinsic properties of presumed motor neurons.
Whole cell recordings were made from twelve presumed motor neurons, identified by size (capacitance) and location (Fig. 4.6C) in the ventral horn (lateral motor pools). From this collection of recordings, neurons with a resting membrane potential greater than -50mV were discarded. To maintain the resting membrane potential at -60mV, sub- threshold changes in potential induced by drug applications were offset by direct current injection. The passive neuron properties of the eight neurons from which recordings were made are listed in Table 2.1. These results describe the collated effects of the NO scavenger PTIO on the eight motor neurons.
The effect of PTIO on the following sub-threshold and supra-threshold intrinsic neuronal properties is reported: input resistance (MΩ); frequency-current (f–I)
relationship (slope – Hz/nA); rheobase current; maximum firing frequency (Hz); firing threshold (mV); maximum rate of rise (V/s); fast AHP and slow AHP; and action potential amplitude (mV).
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Sub-threshold properties
Overall the resting membrane potential did not appear to change with the application of PTIO (50µM); the resting membrane did not change in four cells, though it was marginally hyperpolarised in three cells and slightly depolarised in one cell during application of PTIO (ns; mean ΔRm =3±3mV; n=8).
The input resistance (Rn) of motor neurons was determined before and after addition of
50µM PTIO by measuring the membrane potential change from -60mV in response to the input of incremental, depolarising steps of square current (1s) applied in the sub- threshold range. The mean membrane potential response to each current step was plotted to give the voltage-current (V-I) relationship (Fig.4.6A and B). PTIO caused an increase in the slope of the voltage-current relationship (n=6/8; Fig. 4.6A and B) and did not significantly alter the input resistance of motor neurons.
Supra-threshold properties
Next, the effects of the removal of endogenously produced NO on the frequency versus injected current or f–I relationships of motor neurons was investigated. The mean firing frequency was recorded during a series of incremental steps of depolarising current and the resulting firing frequency was plotted against the injected current.
In four of the eight cells from which recordings were made, an increase in excitability, indicated by the shift of the steady-state frequency-current relationship in an upward direction and to the left hand side, was noted in half of the neurons from which recordings were made (no change, n=4/8 and increase, n=4/8; Fig.4.7A and B). However, PTIO did not significantly change the excitability of motor neurons during repetitive firing as indicated by the slope of the relationship.
During repetitive firing protocols, the additional measures of excitability, rheobase current, and maximum firing frequency were measured in the group of eight neurons. PTIO did not significantly affect rheobase current (the minimum current required to
138 elicit an action potential) and from the frequency-current relationship data no change in maximum firing frequency was noted (Fig.4.7D).
A depolarising current-ramp, analogous to synaptic input, was used to elicit an action potential to determine the change in action potential parameters during control and drug condition. The voltage response to a depolarising current-ramp was used to measure the action potential amplitude and the voltage response to a depolarising current-ramp differentiated to calculate the maximum rate of rise, firing threshold of the first action potential fired (Fig. 4.8A). The maximum rate of rise and action potential amplitude were not significantly affected during PTIO application (Fig.4.8B and D, respectively). The voltage threshold for the firing of an action potential remained unchanged (Fig. 4.8C).
The amplitude of the fast and slow action potential AHP (fAHP and sAHP, respectively) was measured in control and during PTIO application (Fig. 4.9A and B, respectively). Overall, during the application of PTIO, the amplitude of the fAHP and the sAHP were not significantly affected (Fig.4.8A to D, respectively). However, both the fAHP and sAHP decreased in amplitude after PTIO was removed from the perfusate (P<0.05; -43±23%, n=6 and -58±22%, n= 6; Fig.4.9C and D, respectively).
From these data obtained from experiments using the NO scavenger PTIO to remove endogenous NO, it is likely that NO has an excitatory role in the modulation of motor neuron intrinsic properties as demonstrated by the decrease in both fAHP and sAHP, after removal of the NO scavenging agent, PTIO. These data suggest that endogenous NO is involved in the subtle modulation of the intrinsic properties of spinal motor neurons while the locomotor network is quiescent.
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Figure 4.6. The NO scavenger PTIO does not alter the sub-threshold properties of spinal motor neurons. The change inmembrane potential was recorded during Ai Control (aCSF alone), PTIO (50µM added to perfusate) and Wash (aCSF alone) in response to Aii sub-threshold depolarising current steps. B Plot of the V-I relationship of a motor neuron in control, PTIO and wash. C Cell plot indicating location of motor neurons exposed PTIO. D Input resistance was not significantly affected during PTIO application but was significantly reduced during wash (control and PTIO n=8 and wash n=5; P<0.05; asterisk indicates significance).
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Figure 4.7. Endogenous sources of NO do not alter the firing properties of quiescent motor neurons. Ai
Repetitive firing was recorded in Control (aCSF alone), PTIO (50µM added to perfusate) and Wash (aCSF alone) in response to Aii incremental 1s square depolarising current steps (900pA illustrated). B
Firing frequency versus injected current (f-I) relationship exhibits a shift to the left-hand side indicating an increase in excitability during application of PTIO (50µM). C The slope of the f-I relationship was not significantly affected by PTIO at all concentrations (control and DEA NO n=8 and wash n=6). D the current required to elicit a single action potential, rheobase, was not significantly altered during PTIO application (control and PTIO n=8 and wash n=5). E The maximum firing frequency was not significantly affected by application of 50µM PTIO (control and PTIO n=8 and wash n=5).
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Figure 4.8. Removal of endogenous NO does not alter action potential parameters. Ai Action potentials were recorded in Control (aCSF alone), PTIO (50µM added to perfusate) and Wash (aCSF alone) in response to Aii a depolarising ramp of current (1500pA illustrated). B The maximum rate of rise (MRR),
C voltage threshold for action potential generation and D action potential amplitude were not significantly affected by the application of PTIO (control and PTIO n=8 and wash n=5). However, both the MRR and AP amplitude showed trends towards a decrease. A larger sample of neurons is needed to determine whether this trend is significant.
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Figure 4.9. Endogenous NO modulates the action potential AHP. Ai, Bi Action potentials were recorded in Control (aCSF alone), PTIO (50µM added to perfusate) and Wash (aCSF alone) in response to Aii an incremental depolarising square current pulse (320pA illustrated and Bii incremental 1s square depolarising current steps (900pA illustrated). C The fast AHP was not affected by the application of 50µM PTIO but was significantly reduced during washout (control and PTIO n=8 and P<0.05, wash n=6). D The slow AHP was not affected by the application of PTIO but was significantly reduced during washout (control and PTIO n=8 and P<0.05, wash n=6).
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