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J Neurophysiol
2016 Mar 01;1153:1446-57. doi: 10.1152/jn.00283.2015.
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A behaviorally related developmental switch in nitrergic modulation of locomotor rhythmogenesis in larval Xenopus tadpoles.
Currie SP
,
Combes D
,
Scott NW
,
Simmers J
,
Sillar KT
.
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Locomotor control requires functional flexibility to support an animal's full behavioral repertoire. This flexibility is partly endowed by neuromodulators, allowing neural networks to generate a range of motor output configurations. In hatchling Xenopus tadpoles, before the onset of free-swimming behavior, the gaseous modulator nitric oxide (NO) inhibits locomotor output, shortening swim episodes and decreasing swim cycle frequency. While populations of nitrergic neurons are already present in the tadpole's brain stem at hatching, neurons positive for the NO-synthetic enzyme, NO synthase, subsequently appear in the spinal cord, suggesting additional as yet unidentified roles for NO during larval development. Here, we first describe the expression of locomotor behavior during the animal's change from an early sessile to a later free-swimming lifestyle and then compare the effects of NO throughout tadpole development. We identify a discrete switch in nitrergic modulation from net inhibition to overall excitation, coincident with the transition to free-swimming locomotion. Additionally, we show in isolated brain stem-spinal cord preparations of older larvae that NO's excitatory effects are manifested as an increase in the probability of spontaneous swim episode occurrence, as found previously for the neurotransmitter dopamine, but that these effects are mediated within the brain stem. Moreover, while the effects of NO and dopamine are similar, the two modulators act in parallel rather than NO operating serially by modulating dopaminergic signaling. Finally, NO's activation of neurons in the brain stem also leads to the release of NO in the spinal cord that subsequently contributes to NO's facilitation of swimming.
Fig. 1. Developmental increase in spontaneous swimming behavior. A: examples of postembryonic stage 37/38 and larval stages 45 and 47 Xenopus laevis tadpoles. Drawings kindly provided by Laurence D. Picton, with permission. B: bar graph of the displacement during swimming measured over a 5-min period at different larval stages. C: bar graph of percentage of total time spent swimming measured over 5 min at different larval stages. Insets show typical swim trajectories at stages 37/38 [2 days postfertilization (dpf)] and 45 (4 dpf). Values are means ± SE. ns, Nonsignificant. ***P < 0.001.
Fig. 2. Comparison of fictive swimming in Xenopus tadpoles at stages before and after onset of free feeding. Ai: preparation used at stages 37/38–44; schematic shows a stage 42 animal immobilized in α-bungarotoxin with locations of extracellular ventral root (VR) recording electrodes on left (L) and right (R) sides and a tail-skin stimulating electrode (SE). Aii: a single episode of fictive swimming evoked by a 1-ms electrical current pulse applied via the SE. Note that, after the end of the swim bout, the preparation remains silent in the absence of further stimulation. Below is an expanded excerpt during swimming activity highlighting L/R alternation of VR bursts. Bi: equivalent preparation at stage 45–47. Bii: in the absence of any extrinsic stimulation, the preparation spontaneously generates regular episodes of fictive swimming, here, every ∼20 s. Below is an expanded excerpt of swimming activity during the indicated episode again showing L/R VR burst alternation.
Fig. 3. Development of fictive swimming in later stage Xenopus tadpoles. Ai: completely isolated central nervous system (CNS) preparation at pro-metamorphic (48–62) larval stages with recording electrodes (at VR L12, R20). Aii: repetitive bouts of spontaneous fictive swimming. Shown below is an expanded excerpt of fictive swimming and method used to calculate the percentage of time spent active (see also at top). Other measured rhythm parameters within swim episodes, burst duration (BD), cycle period (CP) and episode duration (ED), are also indicated. B: bar graph illustrating the developmental increase in spontaneous fictive swim occurrence expressed as a mean percentage of time (over a 5-min period) that preparations were active. Values are means ± SE. *P < 0.05.
Fig. 4. Developmental switch in nitric oxide (NO) modulation of fictive swimming in Xenopus tadpoles. Ai: extracellular recording (VR R5) from a stage 42tadpole (as in Fig. 2Ai) of evoked episodes of fictive swimming in control, in the presence of the NO donor diethylamine NONOate (DEA-NO; 200 μM) and during drug washout. Aii: pooled data depicting the mean ED in each condition (n = 9). Bi: extracellular record (VR R7) from a stage 47tadpole (as in Fig. 2Bi) of spontaneous fictive swimming occurring in control conditions and when it increased in the presence of DEA-NO (200 μM). Bii: pooled data showing mean percentage of time spent active in each condition (n = 5). Biii: pooled data showing the mean time spent active following co-application of the NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME; 1 mM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 μM, n = 4). *P < 0.05.
Fig. 5. NO donors increase the occurrence of spontaneous locomotor activity at pro-metamorphic stages. A: VR recording from a stage 54tadpole both prior to, and during, NO donor [S-nitroso-N-acetyl penicillamine (SNAP), 200 μM] application and then following washout with expanded excerpts of L/R alternating VR (L/R 16) bursting during fictive swimming in each condition. Bi: pooled data showing the mean percent time spent active in each condition (n = 13). Bii: pooled data from the same preparations [as illustrated in expanded excerpts in A showing mean BD, CP, and ED (see Fig. 3Aii)] relative to control values during and after SNAP application. C: same pooled data analyses as in B, but for application of a different NO donor, DEA-NO (50–200 μM; n = 14). Values are means ± SE. *P < 0.05.
Fig. 6. Scavenging endogenous NO and inhibiting endogenous NO synthase (NOS) activity decreases fictive locomotion occurrence. A: VR recordings (VR L17) from a stage 55tadpole before, during bath application and following washout of the NO scavenger PTIO (50–200 μM). Bi: pooled data of the mean time spent active in control, under PTIO application and during washout (n = 10). Bii: pooled data of the mean percentage of time spent active before, during and after application of the NOS inhibitor l-NAME (1–2 mM; n = 6). C: VR recordings (VR R18) from a stage 55tadpole of locomotor activity following sequential applications of combined PTIO (100 μM) and l-NAME (1 mM), and then NO donor DEA-NO (200 μM). D: pooled data of the mean percentage of time spent active during the drug manipulations in C (n = 4). Values are means ± SE. *P < 0.05. E: during prolonged exposure to PTIO and l-NAME, spontaneous locomotor activity was generally completely abolished (c.f., top traces in Ei and Eii; also see C and D). However, the central pattern generator (CPG) network remained functional during this drug-induced silence since electrical stimulation of the optic tectum (* in Eii) could elicit episodes of rhythmic activity that were very similar from spontaneous fictive locomotion in control (c.f., bottom traces in Ei and Eii).
Fig. 7. NO's effects on fictive locomotor occurrence at pro-metamorphic stages are mediated in the brain stem (BS). A: schematic of the recording set up for split-bath experiments (adapted from Clemens et al. 2012). B: VR (VR R20) recording from a stage 54tadpole showing spontaneous locomotor activity expression during CNS region-specific application of the NO donor SNAP (200 μM). C: pooled data for the mean duration of locomotor activity relative to control (0%) during and after application of NO donors SNAP (200 μM) or DEA-NO (50–200 μM) to the spinal cord (SC) alone (left bar graphs) or the BS alone (right bar graphs) (n = 8). D: same pooled analyses as in C, but for the location-specific co-application of l-NAME and PTIO to either the SC (left; n = 6) or the BS (right; n = 5). Values are means ± SE. *P < 0.05.
Fig. 8. NO's activation of the locomotor CPG via the BS is independent of spinal DA1 receptors, but requires endogenous spinal NO. A: VR recording (VR R18) from a stage 55tadpole showing spontaneous locomotor activity expression during CNS region-specific application of the DA1 receptor antagonist SCH-23390 (SCH; 50 nM), DEA-NO (200 μM), PTIO (100 μM) and l-NAME (1 mM). NB: the regular low-level activity evident in these traces is nonphysiological noise from the Peltier cooling system. B: pooled data of the time spent active during the successive drug applications (n = 6). Values are means ± SE. *P < 0.05.
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