| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
J Physiol (2003), 551.2, pp. 575-587
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.045229
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
The monoamine noradrenaline (NA) can initiate and/or modulate locomotion in a variety of vertebrates. Here we report that exogenous NA application can facilitate two completely different fictive behaviours in embryos of the common frog Rana temporaria, depending on whether spinal networks are connected to supraspinal centres. When the nervous system is intact, NA elicits a non-rhythmic coiling motor response, reminiscent of a spontaneous behaviour appropriate to drive hatching movements, but has only minor effects on evoked swimming activity. After the spinal cord has been severed from the brain, spontaneous coiling is no longer observed, nor can NA elicit it, but the amine can 'release' swimming rhythm generation in response to electrical skin stimulation. The rhythm is similar, but relatively inflexible when compared to fictive swimming recorded from intact animals. Our pharmacological tests indicate that1-adrenoreceptors are involved in the permissive role of NA during spinalised rhythmic swimming and that the fictive coiling response to NA in intact animals involves descending inputs and the activation of
1-adrenoreceptors. Furthermore, the subtle effects of NA on evoked swimming in intact animals were mimicked by either
(Received 14 April 2003; accepted after revision 11 June 2003; first published online 8 August 2003)
Corresponding author K. T. Sillar: School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, Scotland, UK. Email: kts1{at}st-andrews.ac.uk
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The rhythmic movements responsible for vertebrate locomotion are driven by central pattern generators located in the spinal cord (Delcomyn, 1980; Grillner, 1985). One major goal in the pursuit of the spinal mechanisms underpinning locomotion is the recovery of function after injury (Schouenborg & Kiehn, 2002). Therefore, many studies are conducted in which injury is inflicted by either complete or partial spinal cord lesion (Barbeau et al. 1999). Historically, 'fictive' rhythmic activity appropriate to drive locomotor movements has been most successfully restored after complete lesion using pharmacological agents that replace the normal source of descending aminergic inputs (Viala & Buser, 1969; Forssberg & Grillner, 1973; Barbeau & Rossignol, 1990, 1991; Pearson & Rossignol, 1991; Kiehn et al. 1992). However, the same agents can actually have deleterious effects on locomotor recovery in animals with only a partially lesioned spinal cord, abolishing the weight support and stepping movements that can been attained solely through treadmill training (Rossignol et al. 2001). The differential effects of amines during rhythmic movements in intact versus spinalised animals will be important to resolve, because similar anomalies are apparent during pharmacotherapy in human patients (Dietz et al. 1995; Remis-Neris et al. 1999).
Frog embryos can provide useful insights into the location-specific effects of descending aminergic inputs. First, their nervous system is remarkably simple, yet exhibits many of the basic features of mammalian rhythmogenesis (Roberts, 1990). Second, the spinal circuitry responsible for rhythm generation in frog embryos is readily accessible to physiological and pharmacological manipulations. Third, the behavioural repertoire that emerges from this circuitry is limited and easily identified. Finally, there is a considerable body of evidence that aminergic modulation of spinal circuitry occurs in frog embryos (Sillar et al. 1998, 2002). We have chosen to investigate the location-specific effects of noradrenaline (NA) in embryos of the common frog Rana temporaria, primarily because, in contrast to a closely related species, embryos of the clawed frog Xenopus laevis (McDearmid et al. 1997; Fischer et al. 2001; Merrywest et al. 2002), very little is known of the noradrenergic modulation of locomotion in Rana (McDearmid & Sillar, 1997). In addition, there is reason to believe that descending aminergic inputs are more advanced in this species as compared to Xenopus at equivalent developmental stages (van Mier et al. 1986; Woolston et al. 1994). This is based on a comparison of the respective states of spinal serotonergic innervation, which are thought to be instrumental in transforming the relatively inflexible embryonic motor pattern in Xenopus (Kahn et al. 1982) into a more flexible larval one (Sillar et al. 1991, 1992a,b, 1995). Therefore any investigations using Rana are likely to shed light on more mature mechanisms of aminergic interaction with spinal networks. Here, we report that in Rana, NA can also have differing roles depending on the presence or absence of descending inputs. Moreover, these discrepancies can be explained on the basis of pharmacology. Aspects of this work have been published in abstract form (McLean & Sillar, 2001).
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Rana temporaria embryos were obtained during the spring breeding season (ca February to April) from a commercial source (Blades Biological Ltd, Cowden, UK) or collected from local duck ponds and kept in dechlorinated tap water between 4 and 22 °C until at the correct developmental stage (Fig. 1A; Gosner, 1960). All experiments were performed on pre-feeding embryos and are not therefore subject to UK Home Office regulations regarding animal experimentation as described in the Animals (Scientific Procedures) Act 1986.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 1. The experimental set-up A, experiments were conducted using Rana embryos staged according to Gosner (1960). Ba, fictive motor responses were recorded from embryos immobilised in | ||
Electrophysiological recordings
Rana embryos were first anaesthetised in tricaine methanesulphonate (MS-222, 0.1-1 %; Sigma-RBI, Poole, UK) and then scored along the dorsal fin to aid immobilisation by immersion (ca 60-90 min) in the neuromuscular blocker -bungarotoxin (12.5 µM; Sigma-RBI). Once removed from the toxin, the embryos were pinned down through the notochord on their right side onto a rotatable platform covered in silicone elastomer (Sylgard-184, Dow-Corning, Midland, MI, USA) within a 5 ml chamber, through which 100 ml of saline was re-circulating at 5 ml min-1 (composition (mM): 115 NaCl; 2.5 KCl; 2.5 NaHCO3; 10 Hepes; 1 MgCl2; and 2 CaCl2; pH 7.4). The flank skin was then removed from approximately the otic capsule to the anus using fine forceps and finely etched tungsten dissecting needles. For contralateral recordings, the embryo was positioned dorsal side up, secured with etched tungsten pins, and a portion of the flank skin on the right side was also removed (Fig. 1Ba). For spinalisation experiments, the dorsal portion of the second to the fifth myotome (as numbered from the otic capsule) was removed to facilitate access to the spinal cord (Fig. 1Bb), which was then severed from the brain using dissecting needles at the level of the third to the fourth myotome, and the embryos were left to recover for 10-20 min. Suction electrodes (ca 50-70 µm tip diameter), fashioned out of non-filamented borosilicate glass capillaries (1 mm outer diameter; Clarke Electromedical Instruments, Reading, UK) and fine-bore polythene tubing (2 mm outer diameter; Smith Industries Medical Systems Portex Ltd, Keene, NH, USA), were either placed over the intermyotomal clefts to record extracellular ventral root activity or placed on the tail skin to evoke it, with 1 ms electrical current pulses delivered via fine copper wire attached to a DS2 isolated stimulator (Digitimer, Welwyn Garden City, UK; Fig. 1Ba).
Data acquisition and analysis
Electrophysiological data were amplified 
10 000 via a differential AC amplifier (A-M Systems, Sequim, WA, USA) and stored on videotape using a PCM-4/8 adapter (Medical Systems Corporation, Greenvale, NY, USA). Data were either printed as hard copy off-line with an ORP oscillographic recorder (Yokogawa Ltd, Runcorn, UK) or digitised off-line with an A/D interface (Cambridge Electronic Design, Cambridge, UK), and were measured using the Spike 2 data analysis software (Cambridge Electronic Design) and DataView analysis software (courtesy of W. J. Heitler, University of St Andrews, St Andrews, Scotland, UK). Measurable parameters for non-rhythmic coiling activity included the duration of motor bursts, their longitudinal delay along the body and their spontaneous frequency. The burst durations and longitudinal delays were measured during 30 s of light dimming, when fictive coiling activity is most likely to occur (Soffe, 1991), while the spontaneous frequency was measured 5-10 min after drug application. Measurable parameters for rhythmic swimming activity included burst durations and longitudinal delay, but also the time between successive motor bursts (cycle period), the phase of contralateral motor bursts (calculated as the contralateral delay divided by the cycle period) and the total amount of time swimming activity lasted (episode duration). During experiments the amplitude of motor bursts could fluctuate according to the relative seal of the suction electrode, so this parameter could not be reliably analysed. Swimming was elicited by electrical stimulation and three consecutive episodes measured in control, drug and wash conditions with a 60 s rest interval (omitting the first episode due to the confounding effects of differing rest intervals). Single averages were subsequently determined for the measurable parameters per animal, the data were pooled and Student's paired t test was used to test significance from control values at P < 0.05. All statistical analysis was conducted using Microsoft Excel for Windows. Data are reported in the text as means ± S.E.M. A total of 86 Rana embryos were used in this study.
Drugs
The drugs used in this series of experiments were arterenol (noradrenaline, NA; 10-20 µM), clonidine (150-200 µM), isoproterenol (50-100 µM), metaproterenol (50-150 µM), phentolamine (50-100 µM), phenylephrine (150-200 µM) and propranolol (50-100 µM). All drugs were purchased from Sigma-RBI and dissolved fresh daily in distilled water. Drug concentrations were determined from those used effectively in Xenopus embryos and larvae (Fischer et al. 2001; Merrywest et al. 2002) and were washed off by replacement with fresh saline for 15-20 min.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Induction of a non-rhythmic motor pattern by NA in intact preparations
Rana embryos have a very limited behavioural repertoire, which includes rhythmic swimming movements that generate forward propulsion and non-rhythmic coiling movements that do not constitute locomotion (Soffe, 1991). It is believed that the non-rhythmic behaviour could be termed 'hatching', as it occurs spontaneously during the hatching period, precedes rhythmic locomotory swimming behaviour and would be appropriate to drive the strong body flexions needed to free embryos from their substantial, gelatinous egg membranes (Soffe, 1991). Spontaneous fictive motor activity can also be recorded in immobilised Rana embryos, which, like that observed in the freely behaving animal, consists of both swimming activity (29.2 ± 11.0 % of total, n = 52; for total value, see Table 1) and coiling-like activity (70.8 ± 11.0 % of total, n = 52). The fictive coiling activity was therefore more prevalent, particularly during dimming of the experimental bath illumination (Fig. 2Aa). The basic effects of NA have already been described in preliminary form (McDearmid & Sillar, 1997; McDearmid, 1998), but we have repeated them here to allow for a quantitative comparison with the effects of various pharmacological agents. Shortly after the bath application of NA, the frequency of fictive motor activity increased (Fig. 2Ab) for a transient period of time (ca 5-10 min) after which a wash (20 min or more) in fresh saline was required before the response could be repeated (data not shown). The motor activity induced by NA was predominantly coiling activity (90.1 ± 3.9 % of total, n = 9) and was qualitatively indistinguishable from that which occurs spontaneously or in response to light dimming, as it was characterised by prolonged bursts of motor activity, which propagated in a rostrocaudal direction and never appeared to occur synchronously on the opposite side of the body during contralateral recordings (Fig. 2B).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 2. NA elicits a non-rhythmic motor pattern with minimal effects on evoked swimming parameters Aa, recordings from the fourth (L4) and eighth (L8) intermyotomal clefts in a particularly quiescent preparation illustrate how dimming of the bath illumination (between black arrowheads) can trigger bouts of non-rhythmic motor activity (or fictive coiling), which can also occur spontaneously (grey arrowhead). Ab, shortly after the bath application of NA (at black arrow), bouts of predominantly fictive coiling behaviour are elicited for a transient period of time. Dashed lines are to illustrate trace continuity. B, fictive coiling responses elicited by NA on a faster time scale. C, excerpts of evoked swimming taken approximately 500 ms into the episode in both control conditions (Ca) and the presence of NA (Cb) illustrate some of the measurable parameters, including burst durations (BD), cycle periods (CP), longitudinal delay (LD) and the phase of contralateral bursts ( | ||
Given the known modulatory effects of NA in tadpoles of Xenopus laevis (McDearmid et al. 1997), we were surprised to find no significant effects of NA on the parameters of the swimming rhythm in Rana (Fig. 2Ca and b). While there was a clear trend for burst durations, cycle periods and longitudinal delays to decrease, these differences were not statistically significant (Table 1). There was, however, a noticeable increase in the duration of swimming episodes in the presence of NA (Fig. 2D), similar to that described for Xenopus embryos (Fischer et al. 2001). Despite this increase in episode durations, burst durations and longitudinal delays in the presence of NA remained correlated with cycle periods (Fig. 2Ea and b), as they have been described in detail during control conditions (Soffe, 1991).
Facilitation of swimming by NA in spinalised preparations
Thus, in the immobilised but intact animal, NA selectively increased the probability of occurrence of the non-rhythmic coiling behaviour. We next severed the spinal cord from the brain to assess whether the spinal circuitry alone could account for this response to bath-applied NA. When deprived of inputs from descending interneurons following acute spinalisation, Rana embryos do not produce spontaneous motor activity as they normally do, there is no response to dimming of the bath illumination (Fig. 3Aa) and NA did not elicit the coiling activity described above. In a minority of cases (n = 3/10), NA instead initiated an episode of rhythmic swimming activity (Fig. 3Ab). However, by far the most consistent response to NA in spinalised preparations was the initiation of swimming by stimulation of the flank skin following trains of electrical current pulses (n = 10/10; Fig. 3C). Prior to bath application of NA, electrical skin stimulation elicited sustained bursts of motor activity that appeared uncoordinated. Shortly after application of NA, episodes of swimming could be elicited by skin stimulation (Fig. 3B and C), but only for a transient period of time (ca 5-10 min), after which a rest (ca 20-30 min) was needed before swimming could be elicited again (Fig. 3C). Interestingly, this time window appears to mirror that witnessed for the NA response in intact preparations. The swimming behaviour elicited by electrical stimulation was similar to that in spontaneous episodes of swimming and was characterised by a brief acceleration in swimming frequency followed by a gradual deceleration (Fig. 3D). The cycle periods gradually increased as the episode of swimming progressed, excepting a brief period at the beginning of spontaneous episodes, which corresponded to poorly co-ordinated activity (Fig. 3D) that often propagated in a caudorostral direction (see negative longitudinal delay values in Fig. 3Eb). Similar to swimming in intact Rana embryos, spinalised swimming was characterised by a contralateral alternation of motor bursts, as evidenced by a phase value at or near 0.5 (Table 1; cf. Fig. 2 and Fig. 3). However, unlike fictive swimming recorded in intact embryos (cf. Fig. 2), motor burst durations (Fig. 3Ea) and longitudinal delays (Fig. 3Eb) did not correlate with cycle period. Similarly, the cycle periods rarely exceeded 100 ms and burst durations never exceeded 50 ms, in contrast to the case in intact animals (Table 1).
 |
View larger version [in this window] [in a new window] |
|
|
Figure 3. NA facilitates rhythmic swimming in the absence of descending inputs Aa, recordings from the ninth (L9) and eleventh (L11) intermyotomal clefts in an acutely spinalised preparation illustrate that no spontaneous fictive coiling is present, nor can dimming of the bath illumination elicit it (between black arrowheads). Ab, in a minority of cases, shortly after the bath application of NA (at black arrow) a bout of fictive swimming was elicited. Dashed lines illustrate trace continuity. Note that due to the time scale fictive swimming looks like one continuous motor burst. Within this episode are discrete motor bursts as illustrated in B. B, electrically evoked swimming in the presence of NA in spinalised preparations. Shaded grey boxes in the inter-burst intervals are present for illustrative purposes. C, swimming was more reliably elicited by persistent electrical stimulation of the flank skin (artefacts at asterisks) for a transient period of time. Breaks in subsequent episodes represent 60 s rest intervals, except for the last, which represents a 25 min rest interval (at grey arrowhead). L, left side; R, right side. D and E, further quantification of measurable parameters of NA-evoked swimming and electrical stimulus-evoked swimming in the presence of NA with representative data from different animals. Note the differences as compared to intact animals in Fig. 2D and E on identical scales. | ||
Pharmacology of the spinal NA response
The data thus far suggest that NA can broadly affect network excitability, but with two different behavioural outcomes depending on the presence or absence of inputs from descending interneurons in the brain. We next adopted a pharmacological approach to ascertain the receptor subtypes involved in each response. First, we examined the pharmacology of the response to NA when embryos were spinalised, as this was likely to be the simplest scenario. Prior to NA application, electrical stimuli can only elicit sustained, poorly coordinated bursts of motor activity as described above (Fig. 4Aa). When the broad-spectrum adrenergic -receptor antagonist phentolamine was applied (n = 5), the subsequent application of NA could not facilitate the generation of rhythmic swimming movements (Fig. 4Ab). Phentolamine alone had no observable effects (data not shown). However, in the presence of the broad-spectrum adrenergic -receptor antagonist propranolol, and NA (n = 8), rhythmic swimming movements could be generated in response to electrical stimuli (Fig. 4Ac). Again, propranolol alone had no observable effects (data not shown). The properties of fictive swimming facilitated by NA in the presence of propranolol were similar to those in the presence of NA alone; namely, cycle periods did not correlate with either burst durations or longitudinal delays (Fig. 4C). Also, burst durations and cycle periods rarely exceeded 50 and 100 ms (Fig. 4C), respectively, and the frequency of swimming gradually declined during the course of an episode (Fig. 4B). In addition, after approximately 5-10 min, swimming activity could not be elicited (data not shown). We did not, however, attempt to see whether swimming could again be elicited after a rest.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 4. General antagonism of adrenergic Aa, recordings from the fifth (L5) and eighth (L8) intermyotomal clefts in an acutely spinalised preparation illustrate that in control conditions, persistent electrical stimulation of the flank skin (truncated artefacts at asterisks) results in only poorly coordinated motor activity, which in general lasts no more than a few seconds. Dashed lines represent a break in the trace of approximately 500 ms, to illustrate this point. Ab, pre-application of the | ||
These results suggest the involvement of an -adrenoreceptor subtype, but not a -adrenoreceptor, in the facilitation of rhythm generation in the spinal cord by NA. To test this, the 2-adrenoreceptor agonist clonidine and the 1-adrenoreceptor agonist phenylephrine were each bath-applied separately. In the presence of clonidine (n = 4), no amount of electrical stimulation could facilitate swimming (Fig. 5Aa and b). To ensure that this was not a failure to respond on the part of the animal, NA was always applied after washout of clonidine. The subsequent application of NA consistently facilitated swimming after electrical stimuli (Fig. 5Ac). However, bath application of phenylephrine could mimic the noradrenergic response (n = 7; Fig. 5Ba and b). Again, the properties of fictive swimming resembled those elicited by NA (Fig. 5C and D). Notably, in contrast to NA alone (cf. Fig. 3Eb), the longitudinal delay during fictive spinalised swimming in the presence of either propranolol and NA (Fig. 4C) or phenylephrine (Fig. 5D) rarely propagated in a caudorostral direction.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 5. Adrenergic A, recordings from the ninth (L9) and eleventh (L11) intermyotomal clefts in an acutely spinalised preparation illustrate that before (a) and after (b) bath application of the | ||
Pharmacology of the NA response in intact animals
In contrast to the clear -adrenoreceptor mimicry of the effects of NA in spinal preparations, pre-application of phentolamine (n = 6) could not occlude the facilitation of non-rhythmic, coiling activity by NA in intact preparations (Fig. 6Aa, Ba and b). Neither could clonidine (n = 5) nor phenylephrine (n = 6) mimic the effect of NA on the frequency of spontaneous activity (Table 1; Fig. 6Bc). This lends support to the idea that intact descending systems are essential for the proper expression of the fictive coiling response, given the presumed localisation of 1-adrenoreceptors in the spinal cord. Pre-application of propranolol, on the other hand, could occlude the effects of NA on spontaneous activity (Fig. 7Ab and Bb). This therefore implies a role for -adrenoreceptors in the NA coiling response in intact preparations, via a descending interneuronal pathway. In support of this, bath application of the non-specific 1,2-adrenoreceptor agonist isoproterenol could potently mimic the intact NA response (Table 1), increasing the occurrence of non-rhythmic coiling activity (Fig. 6Bd), for a transient period of time (Fig. 6Ac). Furthermore, the 2-adrenoreceptor agonist metaproterenol could not mimic the response to NA in intact animals (Fig. 6Ad and Bd). Taken together, these data suggest an important role for the 1-adrenoreceptor subtype in the selective facilitation of non-rhythmic coiling behaviour by NA.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 6. Adrenergic Aa, recordings from the seventh (L7) and tenth (L10) intermyotomal clefts in an intact animal illustrate that pre-application of the general | ||
 |
View larger version [in this window] [in a new window] |
|
|
Figure 7. Selective adrenergic Aa, recordings from the ninth (L9) and tenth (L10) intermyotomal clefts in an intact animal illustrate an excerpt of fictive swimming taken 500 ms from the beginning of the episode in control conditions. Ab, after 5-10 min in the presence of clonidine, fictive swimming frequency noticeably increases. Ac, this can be readily reversed after a 15-20 min wash in fresh control saline. Ad, in the same animal, subsequent application of phenylephrine for 5-10 min also increases swimming frequency. Ae, again, this is a readily reversible effect. Note that the two drugs were applied to the same animal on this occasion to illustrate the reversibility of these effects. Shaded grey boxes in the inter-burst intervals are present for illustrative purposes. B and C, further quantification of these parameters with representative data from two different animals illustrates that NA in the presence of propranolol (Prop/NA), but not in the presence of phentolamine (Phent/NA), consistently decreases burst durations and cycle periods throughout the course of an episode (downward and leftward shift) without dramatically altering their correlation. Note that phentolamine also occludes the increase in cycles per episode normally witnessed in the presence of NA. | ||
In addition to monitoring the effect of pharmacological agents on spontaneous activity, we also measured their effects on evoked swimming parameters. This was due to the subtle effects of NA on swimming, which could possibly have been clearer in response to selective pharmacological agents. While agonists and antagonists to -adrenoreceptors did not appear to have any significant effects on the swimming rhythm (Table 1), activation of -adrenoreceptor subtypes did (Fig. 7). For example, both clonidine (Fig. 7Ab) and phenylephrine (Fig. 7Ad) significantly (and reversibly, Fig. 7Aa, c and e) decreased burst durations and cycle periods, particularly with respect to one another (i.e. the percentage of cycle period taken up by burst duration or duty cycle; see Table 1). In addition, pre-antagonism of -adrenoreceptors by propranolol unmasked similar effects with the subsequent bath application of NA (Fig. 7Ba-c), effects that were completely occluded by phentolamine (Fig. 7Ca-c). In contrast to these clear effects, those on the longitudinal delay of motor bursts were not significant (Table 1). However, since the separation distance of recording electrodes rarely exceeded four myotomes (ca 1 mm), the relatively short distance could have obscured effects that would have been more apparent if electrodes were placed further apart. Regardless, selective -adrenoreceptor activation appears to modulate the swimming rhythm, in parallel with the inductive effects of NA on spontaneous coiling behaviour via -adrenoreceptors.
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our results illustrate that: (1) the non-rhythmic, coiling motor pattern facilitated by NA via 1-adrenoreceptor activation requires intact descending systems and therefore cannot be explained by the effects of NA on the spinal circuitry alone; (2) NA can 'release' swimming rhythm generation in the transected spinal cord via 1-adrenoreceptors; and (3) fictive swimming in the presence of NA and the absence of descending inputs is less variable, suggesting that a descending influence, susceptible to both 1- and 2-adrenoreceptor modulation, is required to express a more flexible swimming repertoire. We will now discuss possible mechanisms that could account for the observed results.
Descending inputs and motor control
Descending inputs from the brain innervate the spinal cord during development in a spatiotemporal fashion that closely matches the acquisition of mature locomotor capabilities, such as postural control and walking (Vinay et al. 2000). This has led to the proposal that descending inputs are causally linked to the maturation of spinal networks (Sillar, 1994; Nishimaru & Kudo, 2000; Norreel et al. 2003; see however, Branchereau et al. 2002). However, there is evidence that immature neural networks can perform mature motor tasks, and that it is the descending information that changes instead (Le Feuvre et al. 1999). Regardless, supraspinal centres can exert a powerful influence over spinal rhythm generation, initiating, modulating and terminating locomotion (Grillner & Wallen, 2002; Li et al. 2003). Following spinal damage, the extent of spinal lesion will obviously determine the relative contribution of supraspinal centres to locomotion. Any pharmacological agent will therefore activate not only post-synaptic receptors on spinal neurons but also pre-synaptic receptors on the terminals of descending neurons. Such a situation has been proposed to account for the conflicting effects of clonidine on locomotor recovery in partially lesioned versus totally lesioned cats. For example, in chronically spinalised animals, intrathecal injection of clonidine immediately induces hindlimb stepping movements on a treadmill (Chau et al. 1998). When only the ventral or ventrolateral portions of the spinal cord are lesioned, intrathecal clonidine now interferes with the recovery of postural and motor control obtained through prior treadmill training (Brustein & Rossignol, 1999). It has been proposed that the latter is due to autoreceptor activation on the terminals of intact descending noradrenergic neurons and a consequent decrease in endogenous NA release. However, the sheer complexity of the mammalian nervous system makes it difficult to separate central effects from, for instance, proprioceptive feedback, which contributes to the normal motor pattern in cats (Pearson et al. 1998).
It is therefore encouraging that in the frog embryo, where the cellular and synaptic machinery underlying locomotion is far less complicated, different motor responses to NA are also observed, depending on the relative integrity of descending inputs. In intact animals, NA elicits a non-rhythmic motor response via 1-adrenoreceptor activation (Fig. 8A), which requires descending interneurons and corresponds to the earliest behaviour observed during the hatching period (Soffe, 1991). This 'hatching' response appears to be particularly light sensitive, as dimming of the experimental bath illumination can reliably elicit it (see Fig. 2Aa; Soffe, 1991; McLean et al. 2001). One possible source of descending excitation is the pineal eye. The light-sensing ability of the pineal eye and its role in behaviour are well described in Xenopus tadpoles (Roberts, 1978; Foster & Roberts, 1982; Jamieson & Roberts, 2000), where dimming of the illumination elicits swimming via a descending excitatory glutamatergic pathway (Jamieson & Roberts, 1999). Preliminary results suggest the dimming response is more efficacious in the presence of NA (McDearmid, 1998), indicating that NA may facilitate pineal signalling to reticulospinal centres. The facilitation of glutamatergic signalling by NA via -adrenoreceptors is not unprecedented (Gereau & Conn, 1994). It is therefore likely that NA is facilitating glutamate release in the spinal cord, in such a way as to favour the expression of strong, ipsilateral body flexions. This -adrenoreceptor-mediated response dominates any secondary effects on evoked swimming, which are only unmasked during selective -adrenoreceptor activation. Our spinalisation experiments indicate that 1-adrenoreceptors are located on spinal neurons where they increase network excitability, facilitating swimming in the presence of NA but the absence of descending inputs (Fig. 8B). It would appear in the intact animal that this effect is manifested as an acceleration in swimming frequency and an increase in episode duration. However, we cannot completely rule out the involvement of additional 1-adrenoreceptors located on descending neurons. The fact that 2-adrenoreceptor activation accelerated swimming frequency, but that these receptors did not appear to be present on spinal neurons, certainly suggests such interactions with supraspinal centres.
 |
View larger version [in this window] [in a new window] |
|
|
Figure 8. Schematic summary and interpretation of the results A, with the central nervous system intact, NA facilitates hatching behaviour via | ||
An important caveat when conducting any pharmacological investigation is the specificity of the agents used and their affinity for the receptors they bind to, which may differ between species and according to developmental stage (Schmidt & Jordan, 2000). The concentration of drugs used in our study was determined from those used effectively in a closely related amphibian species at an equivalent stage of development (Fischer et al. 2001; Merrywest et al. 2002). While we cannot completely rule out the possibility that agonists were not entirely selective for a particular receptor, the clean separation of behavioural actions seen by the drugs at our concentration range makes this unlikely.
Duelling amines: NA versus serotonin
The often synergistic and complementary actions of different amines on fictive locomotion are well described, perhaps no more so than in tadpoles of the clawed frog Xenopus laevis, in which bath application of serotonin (5-HT; Sillar et al. 1992b; Wedderburn & Sillar, 1994) or NA (McDearmid et al. 1997; Fischer et al. 2001; Merrywest et al. 2002) leads to a strengthening or weakening of swimming, respectively. Both 5-HT and NA primarily accomplish this via opposing modulatory effects on a common pre-synaptic target, namely inhibitory glycine release from commissural interneurons onto motor neurons (McDearmid et al. 1997). The modulatory effects of 5-HT in Rana embryos are also known, where it increases burst durations and slows swimming frequency (Woolston et al. 1994). Our findings further support a role for 5-HT in the expression of a variable and flexible swimming motor pattern in Rana, since spinalised swimming in the presence of NA, but the absence of raphespinal innervation, is considerably more stereotyped. More importantly, however, until now there has been no evidence that NA modulates fictive swimming in Rana. Critically, the effect on fictive swimming mediated by NA is the opposite of that described for 5-HT, which suggests that as in Xenopus, 5-HT and NA have synergistic roles in Rana.
One intriguing possibility suggested by our results is that NA and 5-HT may interact upstream of the spinal cord, in the brainstem. It is known in mammals that the locus coeruleus and the raphe nuclei modulate the levels of activity of one another (Mongeau et al. 1998; Szabo & Blier, 2001; Pudovkina et al. 2002). For instance, NA neurons inhibit their own activity via 2-adrenergic autoreceptors (Svensson et al. 1975) and modulate the activity of 5-HT neurons via excitatory 1-adrenoreceptors (Baraban & Aghajanian, 1980). Similarly, 5-HT neurons can inhibit their own activity (Adell et al. 2002) and that of NA neurons (Charlety et al. 1991) via 5-HT1A receptors. Interactions between the aminergic nuclei would have consequential repercussions on rhythm generation in the spinal cord (Fig. 8C). For instance, if NA was increasing spinal 5-HT release, then autoinhibition of NA release could result in a decrease in 5-HT release. This effect on endogenous 5-HT release could explain why clonidine decreased burst durations and cycle periods. Clearly, detailed anatomical descriptions of the expression patterns of aminergic receptor subtypes are now needed to supplement the present data by clarifying at what levels the amines may interact and thus further elucidate the mechanisms of their location-specific effects.
Conclusions
Our pharmacological tests of the spinalised response to NA in Rana indicate an excitatory role for the 1-adrenoreceptor subtype in the spinal cord, as has been described in the cat (Chau et al. 1998; Lee & Heckman, 1999), neonatal rat (Sqalli-Houssaini & Cazalets, 2000) and Xenopus tadpole (Merrywest et al. 2003). Similarly, they implicate 2-adrenoreceptors in the modulation of locomotion and 1-adrenoreceptors in the initiation of motor activity, two effects that have also been described for NA in these species (Barbeau & Rossignol, 1990, 1991; Kiehn et al. 1999; Fischer et al. 2001). It could be that these pharmacologically distinct roles are determined by the timing during development of descending NA axons, or alternatively the amine could naturally either elicit a coiling response or modulate swimming in parallel throughout larval life, the selection of which would be determined by its release site and the receptors activated. Regardless, our results are potentially very important as they suggest that the amines may actively regulate the release of each other during locomotion, in addition to their respective post-synaptic targets in the spinal cord. These interactions would not be apparent in reduced preparations with the spinal cord isolated, except of course where there were intraspinal sources of amines, such as in the lamprey (Svensson et al. 2001). Therefore, any pharmacological evaluation utilising the intact nervous system will have to consider secondary effects on other aminergic populations (or other descending systems). The simplicity of the tadpole preparation makes this possibility relatively easy to confirm and could shed light on similar mechanisms that may be at work in higher vertebrates.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Adell A, Celada P, Abellán MT & Artigas F (2002). Origin and functional role of the extracellular serotonin in the midbrain raphe nuclei. Brain Res Rev 39, 154-180 | [Medline] |
| Baraban JM , & Aghajanian GK (1980). Suppression of firing activity of 5-HT neurons in the dorsal raphe by alpha-adrenoreceptor antagonists. Neuropharmacology 19, 355-363 | [Medline] |
| Barbeau H, McCrea DA, O'Donovan MJ, Rossignol S, Grill WM & Lemay MA (1999). Tapping into spinal circuits to restore motor function. Brain Res Rev 30, 27-51 | [Medline] |
| Barbeau H , & Rossignol S (1990). The effects of serotonergic drugs on the locomotor pattern and on cutaneous reflexes of the adult chronic spinal cat. Brain Res 514, 55-67 | [Medline] |
| Barbeau H , & Rossignol S (1991). Initiation and modulation of the locomotor pattern in the adult chronic spinal cat by noradrenergic, serotonergic and dopaminergic drugs. Brain Res 546, 250-260 | [Medline] |
| Branchereau P, Chapron J & Meyrand P (2002). Descending 5-hydroxytryptamine raphe inputs repress the expression of serotonergic neurons and slow the maturation of inhibitory systems in the mouse embryonic spinal cord. J Neurosci 22, 2598-2606 | [Abstract/Full Text] |
| Brustein E , & Rossignol S (1999). Recovery of locomotion after ventral and ventrolateral spinal lesions in the cat. II. Effects of noradrenergic and serotonergic drugs. J Neurophysiol 81, 1513-1530 | [Abstract/Full Text] |
| Charlety PJ, Aston-Jones G, Akaoka H, Buda M & Chouvet G (1991). Participation of 5-HT1A receptors in the decrease by serotonin of activation of locus coeruleus neurons by glutamate. C R Acad Sci III 312, 421-426 | [Medline] |
| Chau C, Barbeau H & Rossignol S (1998). Effects of intrathecal 1- and 2-noradrenergic agonists and norepinephrine on locomotion in chronic spinal cats. J Neurophysiol 79, 2941-2963 |
[Abstract/Full Text] |
| Delcomyn F, (1980). Neural basis of rhythmic behavior in mammals. Science 210, 492-498 | |
| Dietz V, Colombo G, Jensen L & Baumgartner L (1995). Locomotor capacity of spinal cord in paraplegic patients. Ann Neurol 37, 574-582 | [Medline] |
| Fischer H, Merrywest H & Sillar KT (2001). Adrenoreceptor-mediated modulation of the spinal locomotor pattern during swimming in Xenopus laevis tadpoles. Eur J Neurosci 13, 977-986 | [Medline] |
| Forssberg H , & Grillner S (1973). The locomotion of the acute spinal cat injected with clonidine I. V. Brain Res 50, 184-186 | [Medline] |
| Foster RG , & Roberts A (1982). The pineal eye in Xenopus laevis embryos and larvae: A photoreceptor with a direct excitatory effect on behaviour. J Comp Physiol 145, 413-419 | |
| Gereau RW IV , & Conn PJ (1994). Presynaptic enhancement of excitatory synaptic transmission by -adrenergic receptor activation. J Neurophysiol 72, 1438-1442 |
[Abstract] |
| Grillner S, (1985). Neurobiological bases for rhythmic motor acts in vertebrates. Science 228, 143-148 | |
| Grillner S , & Wallen P (2002). Cellular bases of a vertebrate locomotor system-steering, intersegmental and segmental co-ordination and sensory control. Brain Res Rev 40, 92-106 | [Medline] |
| Gosner KL, (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16, 183-190 | |
| Jamieson D , & Roberts A (1999). A possible pathway connecting the photosensitive pineal eye to the swimming central pattern generator in young Xenopus laevis tadpoles. Brain Behav Evol 54, 323-337 | [Medline] |
| Jamieson D , & Roberts A (2000). Responses of young Xenopus laevis tadpoles to light dimming: possible roles for the pineal eye. J Exp Biol 203, 1857-1867 | [Abstract] |
| Kahn JA, Roberts A & Kahin SM (1982). The neuromuscular basis of swimming movements in embryos of the amphibian, Xenopus laevis. J Exp Biol 99, 175-184 | [Abstract] |
| Kiehn O, Hultborn H & Conway BA (1992). Spinal locomotor activity in acutely spinalized cats induced by intrathecal application of noradrenaline. Neurosci Lett 143, 243-246 | [Medline] |
| Kiehn O, Sillar KT, Kjaerulff O & McDearmid JR (1999). Effects of noradrenaline on locomotor rhythm-generating networks in the isolated neonatal rat spinal cord. J Neurophysiol 82, 741-746 | [Abstract/Full Text] |
| Le Feuvre Y, Fénelon VS & Meyrand P (1999). Central inputs mask multiple adult neural networks within a single embryonic network. Nature 402, 660-664 | [Medline] |
| Lee RH , & Heckman CJ (1999). Enhancement of bistability in spinal motoneurons in vivo by the noradrenergic 1 agonist methoxamine. J Neurophysiol 81, 2164-2174 |
[Abstract/Full Text] |
| Li W-C, Perrins R, Walford A & Roberts A (2003). The neuronal targets for GABAergic reticulospinal inhibition that stops swimming in hatchling frog tadpoles. J Comp Physiol [A] 189, 29-37 | |
| McDearmid JR, (1998). Noradrenergic control of spinal motor circuitry in two related amphibian species: Xenopus laevis and Rana temporaria. PhD Thesis, University of St Andrews, UK | |
| McDearmid JR, Scrymgeour-Wedderburn JF & Sillar KT (1997). Aminergic modulation of glycine release in a spinal network controlling swimming. J Physiol 503, 1473-1482 | |
| McDearmid JR , & Sillar KT (1997). A slow non-rhythmic motor pattern elicited by both noradrenaline and nitric oxide in embryos of the frog Rana temporaria. J Physiol 504.P, 12P | |
| McLean DL, McDearmid JR & Sillar KT (2001). Induction of a non-rhythmic motor pattern by nitric oxide in hatchling Rana temporaria embryos. J Exp Biol 204, 1307-1317 | [Abstract] |
| McLean DL , & Sillar KT (2001). Location specific roles for noradrenaline in the central nervous system of the frog embryo revealed by spinalisation. J Physiol 537.P, 147P | |
| Merrywest SD, Fisher H & Sillar KT (2002). Alpha-adrenoreceptor activation modulates swimming via glycinergic and GABAergic inhibitory pathways in Xenopus laevis tadpoles. Eur J Neurosci 15, 375-383 | [Medline] |
| Merrywest SD, McDearmid JR, Kjaerulff O, Kiehn O & Sillar KT (2003). Mechanisms underlying the noradrenergic modulation of longitudinal co-ordination during swimming in Xenopus laevis tadpoles. Eur J Neurosci 17, 1013-1022 | [Medline] |
| Mongeau R, Weiss M, de Montigny C & Blier P (1998). Effect of acute, short- and long-term milnacipran administration on rat locus coeruleus noradrenergic and dorsal raphe serotonergic neurons. Neuropharmacology 37, 905-918 | [Medline] |
| Nishimaru H , & Kudo N (2000). Formation of the central pattern generator for locomotion in the rat and mouse. Brain Res Bull 53, 661-669 | [Medline] |
| Norreel J-C, Pflieger J-F, Pearstein E, Simeoni-Alias J, Clarac F & Vinay L (2003). Reversible disorganisation of the locomotor pattern after neonatal spinal cord transection in the rat. J Neurosci 23, 1924-1932 | [Abstract/Full Text] |
| Pearson KG, Misiaszek JE & Fouad K (1998). Enhancement and resetting of locomotor activity by muscle afferents. Ann N Y Acad Sci 860, 203-215 | [Abstract/Full Text] |
| Pearson KG , & Rossignol S (1991). Fictive motor patterns in chronic spinal cats. J Neurophysiol 66, 1874-1187 | [Abstract] |
| Pudovkina OL, Cremers TIFH & Westerink BHC (2002). The interaction between the locus coeruleus and dorsal raphe nucleus studied with dual-probe microdialysis. Eur J Pharmacol 445, 37-42 | [Medline] |
| Remis-Neris O, Barbeau H, Daniel O, Boiteau F & Bussel B (1999). Effects of intrathecal clonidine injection on spinal reflexes and human locomotion in incomplete paraplegic subjects. Exp Brain Res 129, 433-440 | [Medline] |
| Roberts A, (1978). Pineal eye and behaviour in Xenopus tadpoles. Nature 273, 774-775 | [Medline] |
| Roberts A, (1990). How does a nervous system produce behaviour? A case study in neurobiology. Sci Prog 74, 31-51 | [Medline] |
| Rossignol S, Giroux N, Chau C, Marcoux J, Brustein E & Reader TA (2001). Pharmacological aids to locomotor training after spinal injury in the cat. J Physiol 533, 65-74 | [Abstract/Full Text] |
| Schmidt BJ , & Jordan LM (2000). The role of serotonin in reflex modulation and locomotor rhythm production in the mammalian spinal cord. Brain Res Bull 53, 689-710 | [Medline] |
| Schouenborg J , & Kiehn O (2002). The Segerfalk symposium on principles of spinal cord function, plasticity and repair. Brain Res Rev 40, 1-329 | [Medline] |
| Sillar KT, (1994). Synaptic specificity: development of locomotor rhythmicity. Curr Opin Neurobiol 4, 101-107 | [Medline] |
| Sillar KT, McLean DL, Merrywest SD & Fischer H (2002). Fast inhibitory synapses: targets for neuromodulation and development of vertebrate motor behaviour. Brain Res Rev 40, 130-140 | [Medline] |
| Sillar KT, Reith CA & McDearmid JR (1998). Development and aminergic neuromodulation of a spinal locomotor network controlling swimming in Xenopus larvae. Ann N Y Acad Sci 860, 318-332 | [Abstract/Full Text] |
| Sillar KT, Simmers AJ & Wedderburn JFS (1992a). The post-embryonic development of cell properties and synaptic drive underlying locomotor rhythm generation in Xenopus larvae. Proc R Soc Lond B Biol Sci 249, 65-70 | [Medline] |
| Sillar KT, Wedderburn JFS & Simmers AJ (1991). The development of swimming rhythmicity in post-embryonic Xenopus laevis. Proc R Soc Lond B Biol Sci 246, 147-153 | [Medline] |
| Sillar KT, Wedderburn JFS & Simmers AJ (1992b). Modulation of swimming rhythmicity by 5-hydroxytryptamine during postembryonic development in Xenopus laevis. Proc R Soc Lond B Biol Sci 250, 107-114 | [Medline] |
| Sillar KT, Woolston A-M & Wedderburn JFS (1995). Involvement of brainstem serotonergic interneurones in the development of a vertebrate spinal locomotor circuit. Proc R Soc Lond B Biol Sci 259, 65-70 | [Medline] |
| Soffe SR, (1991). Centrally generated rhythmic and non-rhythmic behavioural responses in Rana temporaria embryos. J Exp Biol 156, 81-99 | [Abstract] |
| Sqalli-Houssaini Y , & Cazalets J-R (2000). Noradrenergic control of locomotor networks in the in vitro spinal cord of the neonatal rat. Brain Res 852, 100-109 | [Medline] |
| Svensson E, Grillner S & Parker D (2001). Gating and braking of short- and long-term modulatory effects by interactions between colocalized neuromodulators. J Neurosci 21, 5984-5992 | [Abstract/Full Text] |
| Svensson TH, Bunney BS & Aghajanian GK (1975). Inhibition of both noradrenergic and serotonergic neurons in the brain by the alpha-adrenergic agonist clonidine. Brain Res 92, 291-306 | [Medline] |
| Szabo ST , & Blier P (2001). Effect of the selective noradrenergic reuptake inhibitor reboxetine on the firing activity of noradrenaline and serotonin neurons. Eur J Neurosci 13, 2077-2087 | [Medline] |
| van Mier P, Joosten HWJ, van Reden R & ten Donkelaar HJ (1986). The development of serotonergic raphespinal projections in Xenopus laevis. Int J Dev Neurosci 4, 465-476 | [Medline] |
| Viala D , & Buser P (1969). The effects of Dopa and 5HTP on rhythmic efferent discharges in hindlimb nerves in the rabbit. Brain Res 12, 437-443 | [Medline] |
| Vinay L, Brocard F, Pflieger J-F, Simeoni-Alias J & Clarac F (2000). Perinatal development of lumbar motoneurons and their inputs in the rat. Brain Res Bull 53, 635-647 | [Medline] |
| Wedderburn JFS , & Sillar KT (1994). Modulation of rhythmic swimming activity in postembryonic Xenopus laevis tadpoles by 5-hydroxytryptamine acting at 5HT1a receptors. Proc R Soc Lond B Biol Sci 257, 59-66 | [Medline] |
| Woolston A-M, Wedderburn JFS & Sillar KT (1994). Descending serotonergic spinal projections and modulation of locomotor rhythmicity in Rana temporaria embryos. Proc R Soc Lond B Biol Sci 255, 73-79 | [Medline] |
Acknowledgements
We are very grateful to J. R. McDearmid and H. Fischer for useful discussions. We also thank S. D. Merrywest for critically reading earlier versions of this manuscript and post hoc data retrieval. This work was supported by The Wellcome Trust.
Author's present address
D. L. McLean: Department of Neurobiology and Behavior, Life Sciences Building, SUNY Stony Brook, Stony Brook, NY 11794-5230, USA.
This article has been cited by other articles:
|
|
 |
|
  V. Thirumalai and H. T. Cline Endogenous Dopamine Suppresses Initiation of Swimming in Prefeeding Zebrafish Larvae J Neurophysiol, September 1, 2008; 100(3): 1635 - 1648. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. McLean and K. T. Sillar Metamodulation of a Spinal Locomotor Network by Nitric Oxide J. Neurosci., October 27, 2004; 24(43): 9561 - 9571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. D. Merrywest, D. L. McLean, J. T. Buchanan, and K. T. Sillar Evolutionary Divergence in Developmental Strategies and Neuromodulatory Control Systems of Two Amphibian Locomotor Networks Integr. Comp. Biol., February 1, 2004; 44(1): 47 - 56. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), calculated as the contralateral delay (CD) over the cycle period. L, left side; R, right side. Shaded grey boxes in the inter-burst intervals are present for illustrative purposes. D and E, further quantification of the measurable parameters with representative data from one animal illustrates a comparison between control and NA values. Note that there are more cycles in an episode in the presence of NA, illustrating its effect on episode durations.
) or fictive coiling (
) is graphically represented in bar charts. Normally, spontaneous fictive activity is dominated by fictive coiling activity. Phent, phentolamine; Prop, propranolol; Clon, clonidine; Phee, phenylephrine; Isop, isoproterenol; Meta, metaproterenol.