2.5 Results Summary
2.7.5 Exogenous application of NO mimics the excitatory effect of light
Exogenous NO was shown to increase the occurrence of spontaneous locomotor activity (Fig. 2.8 & Fig. 2.9) in a manner similar to the effect of light on the isolated nervous system (see Chapter 1). There is a well described phenomenon in vascular physiology where NO is liberated from a molecular store following illumination and this in turn leads to ‘photorelaxation’of the endothelial smooth muscle (Furchgott et al. 1961; Martin et al. 1985; Flitney & Megson 2003) (Karlsson et al, 1984; Martin et al, 1985;
168
Figure 2.19 Exogenous NO mimics the effect of light on spontaneous locomotor activity.
A, When the isolated brainstem and spinal cord of a stage 58 tadpole falls silent in the dark,
activity can be restored by the addition of exogenous NO. Rhythmic locomotor activity occured
spontaneously in the light (A & Bi) but was reduced or abolished in the dark (A; also see Fig.
1.14). Subsequent bath application of the NO donor SNAP (200M) restored spontaneous
activity, mimicking the light condition (A & Bii). After exogenous NO was removed,
spontaneous activity was reduced to the pre-drug level in the dark (A). Light sensitivity
remains, however, and the preparation produced spontaneous rhythmic activity again upon re-
illumination (A & Biii). B, Representative episodes of swimming from A illustrate that the
motor bursts were essentially indistinguishable between conditions although there was sign of
rebound following re-illumination (see increased activity in the light in A and extra motor bursts
in Biii). NB – this is the only example of this response where the preparation was active in
control and showed clear light sensitivity.
5min
SNAP
1s
A
169
A
2min 500ms DEA-NOBi
C
ii
iii
-100 -75 -50 -25 0 25 Swim % BD CP ED R el ati ve to valu e in li gh t (% ) *170
Figure 2.20 Light sensitivity persists in the presence of exogenous NO.
A, The isolated nervous system of a stage 54 tadpole exposed to exogenous NO via bath application of the NO donor DEA-NO (200M), retained sensitivity to changes in ambient light level (see Fig. 1.14 for basic phenomenon). In the presence of NO donors, spontaneous locomotor activity was significantly reduced in the dark to 35.06 ± 14.90% of that in the light (100%; A & C) (N=9: 5 in DEA-NO; 4 in 200M SNAP, p = <0.05, paired t-test). B, Representative spontaneous episodes of swimming from A illustrate the effect of illumination on the basic coordination and other properties of motor bursts. Neither BD nor CP or ED were significantly altered by changes in the light conditions (C).
171
Megson et al, 1995; Rodriguez et al, 2003; see Section 2.8.4. for a full description). Therefore, if a similar biochemical process is present in the nervous system it could potentially link increased illumination with increased free NO and an increase in the occurrence of motor activity.
Endogenous NO was already known affect motor activity in the light since both PTIO and L-NAME were able to reduce the occurrence of spontaneous swimming activity in control (lights-on) conditions (Fig. 2.10-12). The next question to answer was could NO increase motor activity independently of the lighting conditions? In a preparation that showed light sensitivity under control conditions (Fig. 2.19A) the subsequent
application of the NO donor SNAP mimicked the lights-on condition, which was reversible upon washout (N=1). Subsequent illumination highlighted the persistence of the light sensitivity following SNAP washout. Furthermore, the swimming was
essentially indistinguishable between control in the light (Fig. 2.19Bi); SNAP in the dark (Fig. 2.19Bii) and wash in the light (Fig. 2.19Biii). While this experiment was only performed on one occassion it demonstrated that exogenous NO could overcome the inhibitory effects of light on the locomotor network.
If NO is actually involved in the pathway linking light sensitivity to motor activity in the isolated nervous system it may be possible to maximally activate the system such that subsequent changes in illumination do not alter the motor activity. During
application of NO donors SNAP (200M; N=4) or DEA-NO (200M; N=5) the light sensitivity of the isolated nervous system was retained (see Fig. 1.14 for basic
phenomenon). In the presence of NO donors spontaneous locomotor activity in the dark reduced significantly to 35.06 ± 14.90% of that in the light (Fig. 2.20A & C; N=9, p = <0.05, paired t-test). During the same conditions, changes in the lighting conditions did not significantly alter either BD, CP or ED (Fig. 2.20C). This result could be explained if the NO and light-mediated pathways controlling motor output worked independently of one another. However, it also does not preclude the possibility that NO is stored and released within the nervous system as it is in the vascular system. The fact that in pharmacologically excited preparations there is a loss of the light sensitivity (Fig. 1.18) potentially provides further support to this possibility (see Section 2.8.4. for further discussion).
172
One experiment not illustrated here but that was attempted multiple times was to compromise nitrergic signalling with co-application of L-NAME and PTIO and then test the light sensitivity of the preparation. The first problem with this experiment was that spontaneous activity was completely abolished under these conditions (see Fig. 2.12Dii for example) and so subsequent investigation of the effect of light was impossible. Several attempts were made to raise excitability via other means –
application of 0Mg2+ saline, dopamine, NMDA and blockade of GABA-A receptors via bicuculline and GABAzine - however under these conditions light sensitivity was lost (see Fig. 1.18 for example). This precluded any attempt to use these methods to boost excitability in order to test light sensitivity following L-NAME and PTIO application. To be clear, in this situation artificially increasing spontaneous activity would reveal a loss of light sensitivity but this would be no different to the same pharmacological manipulation with nitrergic signalling intact. In the future it would be interesting to attempt a similar manipulation but in a split bath set up. For instance, would light sensitivity remain following L-NAME and PTIO application to the brainstem only? Or, if all activity was still abolished, could an excitatory manipulation be made in the spinal compartment which would allow residual light sensitivity to be tested?
173
2.8 Discussion.