Cyclic nucleotide dependent effects of nitric oxide on the locomotor network

NO is known to exert its effects primarily via sGC, catalysing the production of cGMP which then initiates the PKG pathway. NO can also modulate cellular properties by s- nitrosation, a reaction involving the production of reactive nitrogen species that modify thiol functional groups on proteins by addition of –NO (Thomas et al., 2008). As the main regulatory pathway for NO is via sGC, the present study assessed involvement of this pathway in NO-mediated changes in locomotor output.

The PKG partial agonist 8BrcGMP was first used to assess involvement of the cGMP- dependent signalling pathway in NO-mediated effects on the mammalian locomotor CPG. 8BrcGMP significantly affected both the frequency and amplitude of locomotor burst output. These data suggest that cGMP-dependent signalling is involved in the effects of NO on both locomotor burst amplitude and frequency. Further work is needed to ascertain whether or not the 8BrcGMP effect on amplitude observed in these experiments is concentration dependent compared to that of DEA NO.

To investigate further the likely contribution of the NO/sGC/cGMP/PKG pathway to motor network function, the pathway was inhibited using ODQ, an inhibitor of sGC (Zhao et al., 2000). ODQ reduces the ferrous binding site of sGC. On binding to the haem, sGC still exhibits a basal level of activity, although stimulation of cGMP production by addition of exogenous NO is ablated (Zhao et al., 2000). Locomotor burst frequency increased significantly during high concentration ODQ application (200μM) before declining to below control levels; burst frequency did not recover during washout. Burst amplitude significantly decreased in the presence of high concentrations of ODQ, an effect that again could not be reversed by washout of the inhibitor. Although these data suggest involvement of activation of the sGC signalling pathways

111 by endogenous NO, the non-reversible, inhibitory effects of high concentrations of ODQ on both locomotor burst amplitude and frequency suggest that the block of sGC has a profound and detrimental effect on the ability of the network to function.

To mitigate the detrimental effects of high concentrations of the sGC inhibitor and to investigate further the link between NO-mediated modulation of the locomotor network and the sGC pathway, a lower concentration (50μM) of ODQ was applied in combination with DEA NO. Burst frequency again increased during low concentration ODQ application and this increase was reversed by application of DEA NO. The initial increase in frequency as a result of sGC block indicates that a basal sGC activity is involved in maintaining locomotor frequency; whether this is due to endogenous NO production or some other mechanism is not clear. The subsequent reduction in frequency on addition of DEA NO suggests that NO modulates the frequency of locomotor output by cGMP-independent mechanisms or by further stimulation of sGC, though these mechanisms, again, have not been shown in the present study. In the respiratory system, NO modulates inspiratory drive by a cGMP-dependent mechanism and by a cGMP-independent mechanism, possibly peroxynitrite formation (Pierrefiche et al., 2007). To clarify this mechanism, additional experiments would need to be performed; for instance, extending the current protocol to include washout of DEA NO but not ODQ to determine whether burst frequency increases, or using antioxidant application to neutralise peroxynitrite.

The ODQ-mediated block of sGC prevented all DEA NO-mediated effects on locomotor burst amplitude, indicating the involvement of the NO/sGC/cGMP pathway in modulation of motor neuron output. It is not clear why ODQ at lower concentration does not alter locomotor burst amplitude; perhaps this is a result of the complex concentration-dependent relationship between NO production and locomotor output. Despite this, these results confirm for the first time that the NO stimulation of sGC and the subsequent activation of the PKG pathway by cGMP directly modulates motor neuron output.

112 It is also possible that NO inactivates nNOS via a Fe2+-NO intermediate. NO is produced in pulses according to the turnover rate of the reaction to convert L-arginine to NO and L-citrulline (one pulse/140ms) and at high concentrations of NO (during high turnover), the inhibiting Fe2+-NO intermediate stabilises and inactivates the enzyme (Salerno and Ghosh, 2009, Fernhoff et al., 2009). This turnover-dependent inhibition of nNOS (auto-inhibition) can be compared to the high levels of NO used in the present study, taking into consideration that exogenous increases in the concentration of NO will add to the endogenous tone that exists during locomotor activity. This mechanism could explain the changes in locomotor burst frequency and concentration-dependent changes in amplitude observed on addition of DEA NO.

Evidence for a NO-mediated positive feedback mechanism for the control of locomotor frequency has been put forward in the lamprey spinal cord where NO is produced in an activity-dependent manner and facilitates excitation via a presynaptic mechanism (Kyriakatos and El Manira, 2007). Data from the present study might support a negative feedback role for NO in the mammalian locomotor network. It is possible that by retrograde signalling NO could cause the amplification of presynaptic cGMP modulating neurotransmitter release. cGMP-independent inhibition by s-nitrosation of sGC, NMDA receptors or VDCC channels and auto-inhibition of nNOS might also effect a self-regulating negative feedback loop.

3.5 Conclusion

Future investigation into the effects of NO described here must be taken forward in the context of neuromodulation of the network. NO is known to exert its effects by directly activating secondary messenger pathways (sGC/cGMP/PKG; (Denninger and Marletta, 1999, Garthwaite, 2010), modifying protein residues (s-nitrosation) (Choi et al., 2000), and metamodulating the actions of other neurotransmitters such as glycine and GABA (McLean and Sillar, 2002, McLean and Sillar, 2004, Wexler et al., 1998). Subsequent studies in the locomotor preparation must take into account the nature of neuromodulatory interactions to assign definitively a role for NO in the locomotor CPG,

113 as it is not known whether NO is involved in meta-modulation in the mammalian locomotor network.

The divergent activity-dependent effects of NO in the present study may be a result of state-dependent modulation, and theoretically, a mechanism for state-dependent modulation in vivo. The endogenous availability of NO in combination with other neurotransmitters could be the cause of or exacerbate differing effects on the modulation of rhythmic output. For instance, NO enhances the postsynaptic effects of 5HT release at the synapse between Lymnaea cerebellar giant cells (CGC) and B4 motor neurons involved in feeding (Straub et al., 2007). In addition, in a behavioural study, NO inhibits the food intake stimulated by 5HT1A agonists injected into the rat

midbrain raphe (Currie et al., 2011). In spinal motor neurons, 5HT1A receptors have a

postulated role in inhibitory modulation at the motor neuron initial segment while 5HT2

and 5HT7 are involved in initiating locomotion (Heckman et al., 2009). Also in the rat

spinal cord, low concentrations of 5HT facilitate locomotion in the presence of the mGluR1 agonist DHPG, while at high concentrations of 5HT, DHPG disrupts locomotor output (Taccola et al., 2003). The complexity increases with the evidence that endocannabinoids and NO facilitate long-term depression in the lamprey spinal cord via mGluR activation (Kyriakatos and El Manira, 2007). Therefore, a potential modulatory relationship between NO and 5HT is theoretically possible, given that NO interacts with 5HT at invertebrate motor synapses and neuromodulation by metabotropic mechanisms involving pathways modulated by 5HT and NO occurs in both mammalian and non-mammalian vertebrates.

The experiments reported by McLean and colleagues rely on the manipulation of fictive locomotion induced by electrical stimulation, whereas Kyriakatos and colleagues induced fictive locomotion with NMDA alone in their studies in the lamprey. Both NMDA and 5HT are used in the present study to induce locomotion before assessing the role of NO on the resulting locomotor output. If NO-mediated effects depend on the state, level or nature of excitation that initiates and maintains locomotion then modulation in these three systems must be calibrated to accommodate for the state- dependent differences resulting from different methods of inducing locomotor output.

114 In the isolated mouse spinal cord preparation, stimulation of the cauda equina and subsequent generation of locomotion would provide an endogenous-activity model for the study of endogenous NO effects. Likewise, fictive locomotion induced by 5HT or by NMDA alone would enable better comparison with the findings in the lamprey and reveal neurotransmitter specific interactions which have not been reported in the context of NO and spinal locomotor CPG activity.

If a state- or frequency-dependent interaction or action of NO exists in the lumbar spinal cord network, it could be revealed by further experiments manipulating the starting frequency; for example, by altering the concentrations of the neurotransmitters used to induce fictive locomotion.

The data in this chapter show that NO from both exogenous and endogenous sources modulates mammalian spinal locomotor output. Exogenous NO inhibits locomotor burst frequency and this was corroborated by evidence that endogenous NO, as revealed by both inhibitors and scavengers, increased locomotor output consistent with NO having an inhibitory modulatory role modulating excitatory transmission. NO appears to modulate locomotor burst output by mechanisms both dependent and independent of the classical NO/sGC/cGMP pathway.

Exogenous NO modulates locomotor burst amplitude in a concentration-dependent manner, potentiating burst amplitude at low concentrations and predominantly reducing burst amplitude at high concentrations. However, clarification of this concentration- dependent relationship by scavengers of endogenous NO was less clear, complicated by possible side reactions to produce reactive NO derivatives. Care should be taken in future to account for or avoid possible side reactions when designing experiments of this kind. Inhibition of NOS suggests that NO modulates excitatory transmission in the spinal locomotor network. NO appears to modulate locomotor burst amplitude by a cGMP-dependent mechanism, though further work is required to clarify the results detailed here.

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Chapter 4: Nitric oxide-mediated modulation of the intrinsic