Prueba de hipótesis General

Utilising the NO donor, DEA NO, to increase the exogenous level of NO at a range of concentrations significantly reduced the frequency of rhythmic locomotor output produced by the locomotor CPG. Additionally, DEA NO affected the amplitude of motor neuron output in a concentration dependent manner. Low concentrations significantly increased or did not significantly affect motor neuron output (50μM and 100μM DEA NO respectively, Section 3.3.1), while high concentrations appeared to significantly increase and decrease motor neuron output (200μM and 400μM DEA NO respectively, Section 3.3.1). In a subset of these experiments where a high concentration of DEA NO was used to investigate the effects of NO, biphasic modulation of motor neuron output was observed.

154 Consistent with the effects observed as a result of the increase in exogenous NO concentrations, the removal of NO by high concentrations of the scavenger molecules cPTIO and PTIO increased the frequency of locomotor output suggesting that NO may facilitate inhibition in the CPG network. Furthermore, experiments inhibiting NOS using the competitive, irreversible inhibitors L-NAME and L-NNA suggest that NO provides tonic inhibitory influence in the active locomotor network. Utilising the disinhibited preparation, which records the excitatory output from the locomotor network in the absence of inhibition (i.e. pharmacological block of glycinergic and GABAergic signalling using strychnine and bicuculline), DEA NO released NO reduces the frequency of rhythmic bursting indicating that NO is involved in inhibition of excitatory-driven rhythmic bursting in the locomotor CPG network. However, future studies to characterise this inhibitory effect of NO would be best designed in the presence of inhibition to clarify that NO mediated network modulation changes are due to the actions of NO and not due to significant alteration of the network as a result of pharmacological blockade by strychnine and bicuculline.

High concentrations of the scavenger molecules PTIO and cPTIO reduce and augment motor neuron output, respectively (400μM PTIO and 200/4000μM cPTIO, Section 3.3.2). While it appears that the more membrane permeable compound PTIO decreases motor neuron output, the more hydrophilic scavenger cPTIO increases motor neuron output. Scavenging of NO by membrane permeable PTIO results in a decrease in motor neuron output, consistent with an excitatory role for NO at the level of the motor neuron at relatively low concentrations. Scavenging of NO by the less membrane permeable cPTIO results in an increase in motor neuron output, consistent with an inhibitory role for NO at the level of the motor neuron at relatively high concentrations of NO donor used in the present study. If the discrepancy in response is due to the relative membrane permeability of these compounds, it can be concluded that low concentrations of NO, presumably from neurons in close proximity to the motor neurons, facilitate excitatory motor output confirming that while motor neurons do not produce NO, they express the machinery to process the NO as a result of paracrine activity.

155 These seemingly conflicting actions at the level of the motor neuron may be rationalised as a result of scavenging NO at distinctly different sites of action (either intra- or extra- cellularly). The reactive chemistry of the imidazolineoxyl N-oxide compounds is such that nitrate and nitrites are produced as a result of NO metabolism, (Radi, 2004, Kuzkaya et al., 2005, Thomas et al., 2008) with the possibility of further NO mediated reactions taking place. However, it is unlikely that the discrepancy observed in motor neuron response to these compounds is due to differences in nitrate/nitrite production as both PTIO and cPTIO have been shown to react with NO in stoichiometric manner (Akaike et al., 1993). However, based on this pivotal characterisation study, it is not clear whether the additional carboxy side chain which confers the greater hydrophilic nature of cPTIO versus PTIO, is also involved in nitrate/nitrite production as these experiments monitored the production of the conversion of PTIO to PTI and not of nitrate/nitrite production. Furthermore, NO may react with itself and its metabolites to produce peroxynitrite directly, in the presence of high concentrations of NO as well as on addition of cPTIO, especially in this experimental paradigm where O2 is in excess

within the cellular environment.

It is not clear why scavenging NO using cPTIO should result in a reduction of motor neuron output, opposite in sign to the effect observed with higher concentrations of NO. It is possible that the discrepancy is linked to the apparent concentration dependent effects of NO but further work is needed to clarify whether this is truly a result of NO scavenging at different locales, NO involved in both excitatory and inhibitory signalling, or side reaction chemistry of the scavenging molecules and the resulting biological effect of these by products in addition to the true effects of NO. Future studies should include the use of known peroxynitrite generators such as SIN1 to clarify the possible contribution of NO metabolites and generated reactive species in the modulation of locomotor output and/or the use of endogenous antioxidants, uric and ascorbic acid. Further work involving NO signalling will need to rely on manipulation of downstream signalling pathways as the obvious experimental control - application of depleted pharmacological agent (i.e. DEA NO) – may not result in definitive results. DEA NO metabolites include both nitrates and nitrites and any depletion experiment using this agent will need to first ascertain the effect of nitrates and nitrites alone on the

156 locomotor network. The most obvious and ideal pathway for manipulation is the metabolism of cGMP by phosphodiesterases as readily available commercial drugs target this portion of the signalling pathway (e.g. sildenafil). These experiments should be carried out in concert with manipulation of the PKG portion of the signalling pathway to provide substantive evidence of the downstream role of NO without the possibility of desensitising sGC by excessive stimulation with cGMP.The NOS inhibitors L-NAME and L-NNA increase the frequency of locomotor output, reaffirming the inhibitory role for NO in the CPG network. Lending some weight to the possibility that NO scavenging may be complicated by by-product side reactions, inhibition of NOS consistently causes a reduction in motor neuron output. Though these effects are subtle they are statistically significant and could be interpreted to support the metabolism and regeneration of NO from nitrates/nitrites as previously discussed.

As NO is able to modulate synaptic function through coupling to G proteins and the initiation of signalling cascades as well as direct modification of proteins by s- nitrosation, the contribution of the NO/sGC/cGMP pathway was investigated using the partial PKG agonist 8BrcGMP as well as the irreversible sGC inhibitor ODQ. The results show that inhibition of sGC by a high concentration of ODQ results in unrecoverable diminution of lumbar nerve output (Section 3.3.3). Although ODQ is an irreversible inhibitor of sGC, its simultaneous application with the NO donor DEA NO is justifiable in the protocols of the present study as it has previously been reported that at some level, inhibition of sGC NO binding sites occurs preventing NO mediated signalling without disruption to normal cellular function (Fernhoff et al., 2009, Cary et al., 2006, Derbyshire et al., 2010). The inhibition of sGC by a low concentration of ODQ, resulted in an increase in frequency of locomotor output followed by a subsequent decrease in frequency on addition of NO donor, again suggesting that NO is involved in maintaining an endogenous inhibitory tone during locomotor activity.

The amplitude of motor neuron output is not significantly affected by a low concentration of ODQ or by the simultaneous application of ODQ and DEA NO suggesting that the predominant mechanism by which NO mediates its effects in lumbar motor neurons is through the NO/sGC/cGMP pathway. Surprisingly, the partial PKG

157 agonist reduces the amplitude of motor neuron output at the relatively high concentration used in the present study. To clarify whether this is a true conflict or a result consistent with results shown herein – that high concentrations of NO donor reduce the amplitude of motor neuron output – further experiments will need to be conducted using a similar range of concentrations of agonist as donor used in the present study.

Taking into consideration the results from whole nerve recordings in the present study, initially it was not obvious whether NO is involved in maintaining an endogenous inhibitory tone during fictive locomotion by increasing inhibition or by a decrease in excitation. Using the disinhibited preparation, where glycinergic and GABAergic transmission is blocked, I have shown that the NO donor DEA NO produces results consistent with NO causing a reduction in excitation in the CPG network.

Furthermore, donor released NO increases motor neuron output at low concentrations and but does not decrease motor neuron output at high concentrations. These results confirm that the concentration dependent effects observed in the presence of inhibition are mediated by similar mechanisms (i.e. by a reduction in excitation at low concentrations with an inhibitory component revealed at high concentrations). Although it is likely that the excitatory network activated in the absence of inhibition is not identical to that in the standard preparation, these results are still very strongly indicative of an actual role for NO in modulating excitatory transmission.

Whole cell patch clamp recordings made from lumbar motor neurons in the presence of the NO donor DEA NO suggest that NO does modulate the excitability of motor neurons. As a result of NO donor application the firing threshold for action potentials as well as the action potential AHP are significantly reduced. These data suggest that NO increases the excitability of motor neurons, enabling them to fire more readily for a given synaptic input and increasing the ability of motor neurons to regenerate action potentials. Data from the whole nerve recordings indicate that the NO donor increases and decreases the amplitude of motor neuron output at low and high concentrations respectively. Taking into consideration the results obtained from whole cell recordings, it is possible that this NO mediated intrinsic increase in excitability results in increased

158 amplitude of motor neuron output at low concentrations but at higher concentrations, over excitation and thus, excitatory block may account for the decrease in amplitude of motor neuron output. Further data collection using whole cell recordings and targeting NO downstream signalling processes such as sGC conversion of GMP to cGMP (using YC-1) or PKG agonist 8BrcGMP, will confirm these results without the gross and indiscriminate activation of signalling pathways by bath application of NO. Furthermore, selective use of pharmacological agents can be used to confirm the NO mediated effects on firing threshold and action potential AHP. It would be prudent for future investigations to take into consideration the recent demonstration that the Na+K+ATPase is intimately involved in short-term memory of motor performance as well as cardiac function by clarifying the possible involvement of NO (Gan et al., 2012, Zhang and Sillar, 2012)

Although NO clearly modulated both fictive locomotion and individual motor neurons, several questions remain unanswered and I have attempted to identify these in my summary of the results detailed in this thesis. The task of taking forward these findings is to not only clarify the detailed sequence and mechanism of NO mediated effect but also determine the possibility that NO modulates locomotor output within a modulatory hierarchy or by action in concert with other neurotransmitters, as has already been described in the tadpole and lamprey (McLean and Sillar, 2004, Kyriakatos and El Manira, 2007).

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Figure 5.1. Schematic diagram summarizing the findings and possible sites of NO modulation in representative A pre and B postsynaptic neurons in the spinal locomotor circuitry. 1, NMDA receptor activation triggers Ca2+ influx followed by the activation of NOS. 2, Activated NOS catalyses the production of NO from L-arginine and 3, NO binds to and activates sGC initiating the conversion of GMP to cGMP with the subsequent activation of secondary messenger pathways such as PKG. 4, NO is free to diffuse to both intra and extracellular targets (auto and paracrine). 5, NO diffuses across the synapse, 6 activating secondary messenger pathways in the postsynaptic neuron. 7, NO targets not investigated in the present study but of relevance to future studies include direct and indirect activation ion channels as well as protein modifications such as s-nitrosation. Pharmacological agents used in the present study are indicated near their propose site of action in grey text.

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