TÉCNICAS E INSTRUMENTOS DE RECOLECCIÓN DE DATOS

To investigate the potential role of NO-mediated signalling in the control of the spinal locomotor circuitry, the NO donor DEA NO, which will increase exogenous levels of NO, was applied to isolated spinal cord preparations in which fictive locomotion had been pharmacologically induced. Fictive locomotion was induced and maintained by activation of the spinal cord locomotor network using a combination of 10μM 5HT and 5μM NMDA by bath application via the circulating, oxygenated perfusate (Materials and Methods, Section 3.2.2.). Once output had stabilised (qualitative evaluation), approximately 60 minutes after application of 5HT and NMDA, 50μM (Fig. 3.3A and B), 100μM, 200μM or 400μM DEA NO was added to the perfusate for a period of 45 minutes. Rhythmic locomotor-related output was recorded from ventral roots via suction electrodes and the effects of DEA NO measured with any changes expressed as a percentage of control.

The frequency of bursts of locomotor-related activity recorded from ventral roots consistently and significantly decreased (Fig.3.3A, B, C and E), compared to control during the application DEA NO at concentrations of 50μM (P<0.05; -15±4%, n=7), 100μM (P<0.05; -5±3%, n=6), 200μM (P<0.05; -17±5%, n=13) and 400μM (P<0.05; - 38±10%, n=10). Typically at the low concentrations (50/100μM), the decrease in frequency reached a maximum 5-10 minutes after DEA NO was added to the perfusate and gradually recovered to near control levels approximately 5-10 minutes before washout. Burst frequency then remained at control levels for the duration of the drug washout period (Fig 3.3C).

At higher concentrations (200/400μM), the peak decrease in frequency occurred 15-20 minutes after the addition of DEA NO to the perfusate and gradually recovered to near control levels approximately 5 minutes before washout. During washout, frequency remained near control levels for approximately 15 minutes and then appeared to increase for the remaining 30 minutes of wash. After approximately 25 minutes of

94 wash, the frequency increased at 400μM and at 200μM the frequency of output remained elevated above control levels.

The effects of DEA NO on the amplitude of bursts of locomotor-related output recorded from ventral roots were more complicated in comparison to frequency. The effect of DEA NO on burst amplitude varied between concentrations and within different preparations such that increases, decreases and biphasic responses, involving transient increases followed by longer lasting decreases, were observed.

During application of 50μM DEA NO, the amplitude increased and reached a peak in the first 10 minutes and remained elevated above control levels during washout (P<0.05; 50μM, control, +9±4%; Fig. 3.3F). The higher concentration of 200µM DEA NO caused an increase, when compared to control, in the amplitude of locomotor- related bursts in a subset of preparations (P<0.05; 200µM, +16±7%, n=7/13 and 400µM, +19±8%, n=6/10 Fig. 3.3F), additionally, a decrease in amplitude was recorded during the DEA NO applications of 200µM (P<0.05; -15±5%, n=8/13) and 400µM (P<0.05; -24±8%, n=7/10). Significant NO effects on both frequency and amplitude were observed at 50, 200 and 400μM but not at 100μM DEA NO, with peak frequency effects observed at 400μM, the highest concentration used, and a concentration dependent change in amplitude across the concentration range. These DEA NO- mediated effects indicate that exogenous sources of NO modulate burst output from the spinal locomotor circuitry.

Burst coordination was then examined to determine whether or not NO-mediated signalling is involved in regulating the pattern of locomotor output, specifically left/right and flexor/extensor alternation. Strong L2/L2 and L2/L5 alternation (Section 3.2.2. for definitions) was observed in control conditions (n=3). The fidelity of L/R alternation was maintained after the addition of 100μM DEA NO (P<0.05; Fig. 3.4Ai). However, mean vector values for L2/L2 alternation appeared to less uniform around the mean between control and drug conditions but the uniformity remained significant (P<0.05; Fig. 3.4Aii). This is most likely due to the longer L5 burst duration as opposed to a fundamental difference in control bursting between these roots or a DEA NO-

95 mediated effect. However, a larger sample of experiments will need to be conducted to clarify the possibility of a DEA NO-mediated effect.

Next, the effects of DEA NO on inter-burst activity were assessed to determine whether DEA NO modulates general excitation of the entire spinal circuitry or if its modulatory effects are specific to locomotor activity (Fig.3.5A and B). The amplitude of inter-burst activity recorded from ventral roots was not significantly affected by DEA NO application in these experiments, though a small increase in variability of inter-burst amplitude was noted. These data indicate that the NO-mediated effects observed on burst frequency and amplitude are specific to locomotor-related activity.

Previously, it has been shown that NO acts via potentiation of inhibitory transmission (glycinergicand GABAergic transmission) to reduce the duration and increase the cycle period of tadpole swimming (McLean and Sillar, 2004). To investigate whether NO- mediated effects on fictive mammalian locomotion occur via the modulation of excitatory or inhibitory transmission, DEA NO was applied to preparations in which disinhibited, synchronous activity was induced using bicuculline (10μM) and strychnine (1μM) in addition to 5HT (5μM) and NMDA (10μM). During pharmacological block of inhibition, the application of 50μM or 400μM DEA NO (Fig. 3.6A-F) were consistent with the effects seen in the presence of inhibitory transmission during the application of 50μM or 400μM DEA NO. DEA NO significantly decreased the frequency of disinhibited rhythms at concentrations of both 50μM (P<0.05; -34±4%, n=5) and 400μM (P<0.05; -59±12%, n=4; Fig. 3.6C and E). The decrease in frequency reached a maximum 15-20 minutes after the addition of 50μM and 400μM DEA NO to the perfusate and recovered, returning to near control levels after approximately 15 minutes of washout.

The application of a low concentration of DEA NO caused an increase in the amplitude of locomotor-related bursts recorded in disinhibited preparations (P<0.05; 50μM, +18±15%, n=5). Amplitude continued to increase for the duration of DEA NO application and remained elevated above control levels but did not appear to increase

96 further during washout (Fig. 3.6D and F). In contrast, high concentrations of DEA NO (400μM) did not significantly affect locomotor burst amplitude

Given that the application of DEA NO to disinhibited spinal cord preparations resulted in changes to both frequency and amplitude, consistent with those that were observed in the presence of inhibition at low donor concentrations, the main effects of NO-mediated signalling on the mammalian locomotor circuit are likely to involve the modulation of excitatory interneurons and/or excitatory synaptic transmission. A role for NO in the modulation of inhibitory transmission is still feasible considering that DEA NO has no effect on disinhibited rhythmic spinal activity.