Electrical coupling synchronises spinal ... - Wiley Online Library

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J Physiol 587.18 (2009) pp 4455–4466

Electrical coupling synchronises spinal motoneuron activity during swimming in hatchling Xenopus tadpoles Hong-Yan Zhang1 , Wen-Chang Li2 , William J. Heitler1 and Keith T. Sillar1 1 2

School of Biology, University of St Andrews, St Andrews KY16 9TS, Scotland, UK School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK

The role of electrical coupling between neurons in the swimming rhythm generator of Xenopus embryos has been studied using pharmacological blockade of gap junctions. A conspicuous effect of 18β-glycyrrhetinic acid (18β-GA) and carbenoxolone, which have been shown to block electrical coupling in this preparation, was to increase the duration of ventral root bursts throughout the spinal cord during swimming. The left-right coordination, the swimming frequency and the duration of swimming episodes were not affected by concentrations of 18β-GA which significantly increased burst durations. However, the longitudinal coupling was affected such that 18β-GA led to a significant correlation between rostrocaudal delays and cycle periods, which is usually only present in older larval animals. Patch clamp recordings from spinal motoneurons tested whether gap junction blockers affect the spike timing and/or firing pattern of motoneurons during fictive swimming. In the presence of 18β-GA motoneurons continued to fire a single, but broader action potential in each cycle of swimming, and the timing of their spikes relative to the ventral root burst became more variable. 18β-GA had no detectable effect on the resting membrane potential of motoneurons, but led to a significant increase in input resistance, consistent with the block of gap junctions. This effect did not result in increased firing during swimming, despite the fact that multiple spikes can occur in response to current injection. Applications of 18β-GA at larval stage 42 had no discernible effect on locomotion. The results, which suggest that electrical coupling primarily functions to synchronize activity in synergistic motoneurons during embryo swimming, are discussed in the context of motor system development. (Received 2 April 2009; accepted after revision 20 July 2009; first published online 27 July 2009) Corresponding author K. T. Sillar: University of St Andrews, Bute Medical Buildings, School of Biology, St Andrews KY16 9TS, UK. Email: [email protected] Abbreviations CBNX, carbenoxolone; CPG, central pattern generator; 18β-GA, 18β-glycyrrhetinic acid; hdIN, hindbrain descending interneuron; R-C, rostro-caudal.

Electrical synapses provide rapid bidirectional pathways for information flow between cells. Many neurons in the vertebrate CNS are coupled by electrical synapses (reviewed in, e.g. Connors & Long, 2004), which often function to synchronize activity in populations of neurons with similar roles (Fuentealba et al. 2004; Leznik & Llinas, 2005; Sinfield & Collins, 2006; Hinckley & Ziskind-Conhaim, 2006; Wilson et al. 2007). Since motor activity often requires tight coordination between motoneurons it is perhaps not surprising that electrical coupling has been widely reported within spinal motor networks (Perrins & Roberts, 1995a,b; Kiehn & Tresch, 2002; Hinckley & Ziskind-Conhaim, 2006; Wilson et al. 2007; Li et al. 2009). The fact that in many systems electrical coupling can be modulated by a variety of neurotransmitters (e.g. Dowling, 1991; Roerig & Sutor, C 2009 The Authors. Journal compilation C 2009 The Physiological Society

1996) raises the possibility that the strength of electrical synapses in motor systems can be regulated under different behavioural circumstances and/or during development (e.g. Sutor & Hagerty, 2005). Swimming in hatchling embryos of the frog Xenopus laevis (stage 37/38) is produced by a central pattern generating (CPG) network distributed throughout the brainstem and spinal cord (Li et al. 2006). During fictive swimming in immobilized embryos, the motoneurons innervating the segmented axial swimming muscles discharge in a characteristic pattern in which firing alternates across the spinal cord and propagates rostrocaudally with a brief delay which is not correlated with cycle period (Tunstall & Sillar, 1993). Motoneurons and premotor interneurons fire only one impulse per cycle of swimming, despite the fact that the same DOI: 10.1113/jphysiol.2009.173468

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neurons are capable of discharging multiply in response to depolarizing injected current pulses (Sautois et al. 2007) or during struggling (Soffe, 1993; Li et al. 2007). During swimming, the single impulse per cycle of homonymous motoneurons exiting a given ventral root is tightly synchronized, leading to brief burst durations of around 5–10 ms and occupying approximately 15% of the cycle period (Sillar et al. 1991). This embryonic motor pattern is superseded early in larval life (stage 42), by one in which the ventral root bursts increase in duration up to 20 ms and occupy up to 50% of a swim cycle (Sillar et al. 1991). Individual neurons now fire multiply in each cycle (Sillar et al. 1992), and the rostrocaudal delay correlates with cycle period (Tunstall & Sillar, 1993). In the present paper we have investigated the role of electrical coupling between neurons in Xenopus embryos (Perrins & Roberts, 1995a,b) by using gap junction blockers. A consistent effect was a pronounced increase in the duration of motor bursts and the development of a correlation between cycle periods and rostrocaudal delays during swimming. Both of these changes are characteristic of swimming at larval rather than embryonic stages. We therefore propose that electrical coupling normally synchronizes homonymous motoneuron firing during swimming in Xenopus embryos, and that during development a decrease in electrical coupling causes de-synchronization and a change in rostro-caudal coordination. The effect of gap junction block in embryos is therefore to mimic some of the processes that occur naturally during the development from embryo to larva. Consistent with this idea gap junction blockade had no discernible effect at the larval stage.

Methods Animals

Xenopus laevis embryos and larvae around the time of hatching (stages 37/38 and 42; Nieuwkoop & Faber, 1956) were obtained by induced breeding of pairs of adults selected from a laboratory colony and injected with human chorionic gonadotropin (1000 U ml−1 ; Sigma). Eggs were collected and reared in aerated trays at temperatures of approximately 17–23◦ C to stagger their development until they had reached the desired stage. All experiments comply with UK Home Office regulations and have been approved by the University of St Andrews Animal Welfare Ethics Committee.

Electrophysiology

Tadpoles were briefly anaesthetized with 0.1% MS-222 (3-aminobenzoic acid ester; Sigma, Gillingham, UK) and

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the trunk skin gashed to facilitate immobilization in 12.5 μM α-bungarotoxin (Sigma) saline, and then pinned in a bath of saline (in mM: 115 NaCl, 3 KCl, 2 CaCl2 , 2.4 NaHCO3 , 1 MgCl2 , 10 Hepes, adjusted with 4 M NaOH to pH 7.4). One or both sides of the trunk skin overlying the myotomal muscles were removed. Extracellular recordings of fictive swimming were made with suction electrodes from ventral roots at intermyotomal clefts. The dorsal parts of rostral myotomes were freed from the spinal cord and the roof of the hindbrain was opened to the neurocoel to improve drug access and access for patch clamp electrodes. Extracellular signals were amplified using differential AC amplifiers (Model 1700, A-M Systems Inc., Carlsborg, WA, USA), displayed on a Gould digital oscilloscope (20 MHz (DSO) 1604), digitized using a CED micro 1401 and stored and processed on a personal computer using Spike2 software (v. 3.21, Cambridge Electronic Design, Cambridge, UK). Simultaneous recordings were made from up to three ventral roots, normally located rostrally at the 4th and more caudally at the 11th or 12th post-otic clefts on the left side and at the 6th–8th cleft on the right side. Fictive swimming was initiated either by dimming the illumination or by stimulating through a glass suction electrode placed on the tail skin, which delivered a 1 ms current pulse via a DS2A isolated stimulator (Digitimer, Welwyn Garden City, UK). Whole-cell patch clamp recordings in current clamp mode were made using electrodes pulled on a Narishige PP830 pipette puller from borosilicate glass capillaries (Harvard Apparatus Ltd). Patch pipettes were filled with 0.1% neurobiotin in the intracellular solution (in mM: 100 potassium gluconate, 2 MgCl2 , 10 EGTA, 10 Hepes, 3 Na2 ATP, 0.5 NaGTP adjusted to pH 7.3 with KOH) and had resistances of ∼10 M. Recordings in whole-cell mode were amplified with an Axoclamp 2B amplifier and digitized using a CED micro 1401. All signals were displayed and saved on a PC using Spike2 software. Neuronal anatomy was revealed as described previously (Li et al. 2002, 2004). All reagents were obtained from Sigma or Tocris Bioscience.

Gap junction block

The specificity of a range of gap junction blockers (carbenoxolone (CBNX), heptanol, flufenamic acid and 18β-glycyrrhetinic acid (18β-GA; Davidson & Baumgarten, 1988)) has already been tested between pairs of hindbrain excitatory descending interneurons (hdINs; Li et al. 2009). The tests identified 18β-GA as an effective electrical coupling blocker with the fewest side effects (Li et al. 2009). 18β-GA was therefore chosen to investigate in more detail the effects on swimming, but we also tested the effects of CBNX, a derivative of 18β-GA and a widely C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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Electrical coupling coordinates locomotor activity

Figure 1. The effects of gap junction blockers on swimming bursts and the electrical coupling of hindbrain dINs of Xenopus embryos Aa, Xenopus tadpole at stage 37/38, from Nieuwkoop & Faber (1956). Ab and c, effects of two gap junction blockers, 18β-GA (90 μM) and CBNX (100 μM), and d, a time control, on ventral root bursts during fictive swimming. B, the increase in ventral root burst durations in 18β-GA correlates temporally with the blocking of electrical coupling between hdINs. Ba, ventral root burst duration measured on the 10th cycle at the beginning of each swimming episode after applying 40 μM 18β-GA; grey: individual measurements from 6 preparations; black: mean durations ± S.D.; Bb, normalised average electrical coupling coefficients (same preparations as Ba) after 18β-GA applied at time zero (see Li et al. 2009). Bc, burst durations measured on the 10th cycle at the beginning of each swimming episode do not change with time under control conditions; grey: individual measurements from 5 preparations; black: average durations ± S.D. Bd, simultaneous whole-cell patch recordings of coupled dINs in control (left column) and after 18β-GA treatment (right column). Upper trace: one example of the 10th ventral root bursts from the beginning of swimming before and after 18β-GA blockade; lower traces: responses of two coupled dINs to hyperpolarizing current injected in either cell. Recording time points of control and block examples were indicated by left and right arrows in Ba, respectively.

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used gap junction blocker, to assess whether its effects on swimming were similar to 18β-GA. An example of the effects of 18β-GA on electrical coupling between hdINs is shown for illustrative purposes in Fig. 1 according to the method described in Li et al. (2009). Time controls

The block of electrical coupling by 18β-GA takes around 35 min to become fully established (Fig. 1Bb) and within the time course of these experiments was irreversible. Therefore two types of control were used in these experiments. First we compared the pre-treatment and treatment conditions within a single preparation, and second we compared a treated preparation with a non-treated preparation maintained for a similar period in control conditions. Data analysis

Electrophysiological data were analysed using Dataview software (v 6.1 pre-release, courtesy of Dr W. J. Heitler), and then all raw data were imported into Microsoft Excel spreadsheets where data from each experimental period were statistically analysed using GraphPad Instat 3.06 software (GraphPad Software Inc.). Means of each condition were compared using Student’s t-test (paired where appropriate), one sample t test (for normalized data), Friedman’s test or Dunnet’s test, and shown as means ± S.E.M. unless stated otherwise. Ventral root bursts were detected as follows. The extracellular recordings were de-meaned (offset removed so mean = 0) and rectified. A threshold cursor was set at the voltage level of the maximum noise in a quiescent part of the recording, and all periods where the voltage crossed this level were marked as events. The Poisson surprise method (Leg´endy & Salcman, 1985) was used to merge appropriate events and detect bursts. Occasional

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outliers were corrected manually. The rectified traces were smoothed with a Gaussian filter set with a −3DB cut off frequency of 50 Hz, and the ‘centre of gravity’ of each burst was detected. The centre of gravity was defined as the time at which the area under the smoothed trace from the start of the burst to that time was equal to the area from that time to the end of the burst. The cycle period was defined as the time from the start of one burst to the start of the next.

Results Gap junction block increases ventral root burst durations

It has been shown previously that neighbouring spinal motoneurons in hatchling Xenopus tadpoles are electrically coupled (Perrins & Roberts, 1995a). To investigate the role of this coupling in coordinating activity during fictive swimming we applied pharmacological blockers of gap junctions. The most conspicuous effect of gap junction block by 18β-GA was to produce a profound increase in the duration of ventral root motor bursts during swimming (Fig. 1Ab, Ba and Bd). There is a strong temporal correlation between the increase in burst durations in the presence of 18β-GA and the decrease in coupling between pairs of hdINs in the hindbrain (Li et al. 2009; Fig. 1Ba, b and d), which suggests that a decrease in coupling at some level in the system may be casual to the increase in burst durations. The changes in burst duration and coupling coefficient took around 35 min to become established after 18β-GA application (Fig. 1Ba, b and d). CBNX had qualitatively the same effects as 18β-GA, namely a time-dependent increase in ventral root burst durations (Fig. 1Ac), consistent with block of gap junctions. However, in addition to these effects presumed to have resulted from decreased electrical coupling, CBNX has been reported to exert a range of side effects on tadpole

Figure 2. Dose-dependent differentiation of 18β-GA effects on fictive swimming of Xenopus embryos In each preparation (n = 7) the concentration was maintained at the specified level (0, 15, 30, 90 μM) for 30 min, and then increased to the next higher concentration. Significant effects on ventral root burst duration (A) occur at lower concentrations (30 μM) than effects on swimming episode duration (B; 90 μM) and swimming cycle period (C; 90 μM). ∗ P < 0.05; ∗∗ P < 0.01. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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neurons (Li et al. 2009). For this reason the present study used 18β-GA as the most selective gap junction blocker in this system. When preparations were maintained for an equivalent period of time in control conditions there were no significant changes in burst durations (Fig. 1Ad and Bc), confirming that the increase is due to the drug application. Dose-dependent differentiation of gap junction block targets

Gap junction block with 18β-GA had a significant effect on burst durations at concentrations as low as 30 μM (n = 6; Fig. 2A), but at this concentration there was no effect on episode durations (Fig. 2B) or cycle periods (Fig. 2C). Increasing the concentration of the blocker to 90 μM, the highest concentration tested, produced an

even stronger effect on burst durations, but now also produced a dramatic and highly significant reduction in episode durations and a small but highly significant increase in cycle periods. Preparations maintained in control conditions for similar periods of time showed no significant change in any of these parameters (n = 5; data not shown). This concentration-dependent differentiation between effects on ventral root bursts and episode durations suggests two different targets, perhaps occurring at different loci in the swimming network (see Discussion). At the lower concentration of 18β-GA (30 μM) the lack of effect on either the mean cycle periods attained during swimming or the duration of entire episodes of swimming suggests that effects on burst duration were mediated primarily at the level of the motoneurons. However, at the higher concentration (90 μM) all three parameters of the swim

Figure 3. Distributed effects of a low concentration of 18β-GA (30 μM) on fictive swimming A, example of ventral root recordings in pre-treatment control (a) and 30 min after 18β-GA application (b). L4 and L11: recording from the fourth and eleventh intermyotomal cleft on left side, respectively; R8: recording from the eighth cleft on right side. B–D, white bars = time 0 (pre-treatment); shaded bars = +30 min (either treatment or control). B, control data (n = 5) and effects of 18β-GA (n = 7) and CBNX (n = 6) on swimming burst durations. a and b: rostral (L4); c and d caudal (L11) burst durations. Asterisks indicate significant differences between time 0 and 30 min. C, 30 μM 18β-GA did not affect left–right coupling. D, the S.D. of the rostro-caudal (R-C) delay was constant over time in control conditions, but was increased by 18β-GA and CBNX. Normalized S.D. of R-C delay was calculated by TS.D. /PTS.D. , where PTS.D. and TS.D. are S.D. of pre-treatment and treatment, respectively. Dotted line indicates the normalized pre-treatment level (i.e. 1) of each experiment. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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motor programme were significantly altered, indicating effects on both motoneurons and premotor rhythm generating interneurons.

Distributed effects of gap junction block on swimming activity

The effects of 18β-GA on fictive swimming were distributed throughout the spinal cord. Thus when simultaneous ventral root recordings were made from multiple locations (Fig. 3A), a highly significant increase in burst durations occurred not only on the left and right sides, but also on caudal as well as rostral ventral roots (n = 7), although the caudal effect was less pronounced (Fig. 3B). This longitudinal difference in effect may be related to the fact that the developmental acquisition of bursting itself follows a rostrocaudal path (see Discussion). 18β-GA did not obviously affect the left-right coordination and the phase relationships between the ventral root bursts on the two sides remained in strict antiphase (Fig. 3C).

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In control conditions the rostrocaudal delays were similar to those reported previously in this preparation (Tunstall & Roberts, 1991); they varied little from one cycle to the next and there was no clear relationship between the duration of the delay and the cycle period (Fig. 4, pre-treatment). Thus, unlike many other swimming animals, including swimming at stage 42 in Xenopus (Tunstall & Sillar, 1993), the delays do not scale with the cycle periods and phase constancy is not maintained. In the presence of 18β-GA the delays between bursts recorded rostrally relative to caudal locations became significantly more variable, an effect that was also observed in the presence of CBNX (n = 6; Fig. 3D). In addition, a small but significant positive correlation between delays and cycle periods emerged in the presence of 18β-GA (Fig. 4). No such positive correlation developed in preparations maintained in control conditions for similar times (n = 5, data not shown). The scaling between delays and cycle periods induced by gap junction block in embryos is qualitatively similar to that occurring normally in larvae (stage 42; Tunstall & Sillar, 1993), which may indicate that a developmental reduction in electrical coupling contributes to the scaling of larval cycle periods and delays (see Discussion).

Effects of gap junction block on motoneuronal firing during swimming

Figure 4. The relationship between R-C delay and cycle period of embryo swimming was changed by 18β-GA A, plot of R-C delay versus cycle period pre-treatment (filled circles) and after 18β-GA application (open circles) for a single preparation. Dashed and continuous lines represent the linear fit of pre-treatment and treatment data, respectively. B, averaged linear fit of pre-treatment (dashed line) and 18β-GA treatment data (continuous line). 18β-GA induced a significant (n = 7; P < 0.05) anticlockwise shift.

The increase in the duration of ventral root motor bursts during swimming in the presence of 18β-GA could be explained either by the de-synchronization of single motoneuron action potentials in each cycle of swimming, or by a transition to multiple action potentials per cycle, or a combination of the two. To test between these two possibilities patch clamp recordings were made from motoneurons, which were subsequently identified following injection of neurobiotin (e.g. Fig. 5C). In seven preparations 18β-GA was applied at the higher concentration of 90 μM in order to speed the onset and maximize the magnitude of effects (n = 7; Figs 5–8), but in an eighth preparation similar motoneuronal effects were also observed at 30 μM (not illustrated). The reliable one-spike-per-cycle firing pattern of motoneurons during swimming (Fig. 5A) did not change after drug application (Fig. 5B), despite the fact that episode durations decreased and ventral root burst durations increased, as described above. However, the variability of motoneuron spike timing increased significantly under 18β-GA. In control conditions the peak of the intracellular spike always occurred just before the onset of the ventral root burst recorded more caudally (Fig. 5Ab), but after 18β-GA application the spike peak could occur at variable times, before, during or near the end of the ventral root burst (Fig. 5Bb, spikes 1, 2 and 3, respectively). This variability C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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in spike timing results from a variable delay in relation to the underlying excitatory drive (e.g. Fig. 5Bb, 1 vs. 3). The variability becomes clearer when many consecutive cycles are triggered off the intracellular spike and the traces are superimposed (Fig. 6Aa, cf. Ba), and when the timings are plotted on a histogram (Fig. 6Ab cf. Bb). Both the average delay and the standard deviation (S.D.) of the delay between the intracellular spike peak and the centre of gravity of the ventral root burst (see Methods) increased in 18β-GA (Fig. 6Ca and b). The alteration in the timing of motoneuron firing in the presence of 18β-GA was accompanied by an increase in spike duration (Fig. 6Ba), which could be due to changes in the electrical properties of individual motoneurons (see below).

Effects of gap junction block on motoneuronal properties in quiescent preparations

The preceding data suggest that the increase in ventral root burst durations after gap junction blockade is caused by a de-synchronization of the discharge of singly firing motoneurons, rather than a transition to multiple firing which was previously curtailed by gap junction coupling. To explore further the effects of gap junction block on neuronal membrane properties, we examined the effects of 18β-GA on motoneurons in the quiescent preparation (i.e. in intervals between swim episodes; Figs 7 and 8). 18β-GA did not significantly affect the resting membrane potential of motoneurons

Figure 5. The effect of 18β-GA (90 μM) on spinal motoneuron activity A and B, example of motoneuron activity during fictive swimming. A, pre-treatment; B, after ca 30 min in the presence of 18β-GA. Aa and Ba, motor activity during entire episodes of fictive swimming; upper trace, motoneuron patch recording; lower trace, ventral root. Note the reduced episode duration in 18β-GA (Ba vs. Aa). Ab and Bb, excerpts of activity from mid-episode at expanded time base to show motoneuron spike timing in relation to the ventral root burst (dotted lines). 1, 2 and 3 indicate different timings of spike peaks in relation to ventral root burst in that cycle. C, camera lucida drawing of neurobiotin-stained motoneuron whose activity is shown in A and B. Dashed lines indicate outline of spinal cord; peripheral axon projects ventrally into the myotome. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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(Fig. 7Aa), nor did it significantly affect the frequencies of spontaneous postsynaptic potentials (Fig. 7Ab). These spontaneous potentials were depolarizing at the resting membrane potential but may have included a mixture of excitatory and inhibitory potentials. The size of the voltage responses of motoneurons to constant amplitude hyperpolarizing current pulses increased in the presence of 18β-GA (Fig. 7B), consistent with an increase in input resistance following closure of electrical synapses. The amplitudes of spontaneous postsynaptic potentials increased after gap junction block, but the effect was not significant (not illustrated). The apparent lack of effect on the amplitudes of spontaneous potentials, despite an increase in membrane resistance, may be a reflection of the electrotonic distances of synaptic input sites and points of electrical coupling relative to the recording site at the soma. There were no consistent effects on the firing properties of motoneurons: in control conditions motoneurons would fire once at threshold and a burst of action potentials with stronger current pulses (Fig. 8Aa), and this continued in the presence of 18β-GA (Fig. 8Ab).

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However, the spike width in response to depolarizing current steps at threshold increased significantly in the presence of 18β-GA (Fig. 8B), although the change was not as large as that which occurred during swimming (Fig. 8C). Neither the amplitudes of motoneuron action potentials nor the spike threshold changed significantly after blocking gap junctions.

Lack of effect of gap junction block on swimming at larval stage 42

The preceding data on the effects of gap junction block at hatching stage 37/38, in which burst durations increase to levels not seen in normal swimming until stage 42 (Fig 9Aa and b upper trace), support the proposal that a decrease in electrical coupling is responsible in part for the development of the ‘bursty’ larval swimming rhythm. If so then gap junction blockers applied at the later stage 42 should have little or no effect on swimming. Indeed neither 18β-GA (90 μM; n = 6) nor CBNX (100 μM; data

Figure 6. The effect of 18β-GA (90 μM) on the timing of individual motoneuron spikes relative to the VR burst during fictive swimming A, pre-treatment episode; B, ca 30 min after 18β-GA. Aa and Ba, 25 superimposed spikes and VR bursts aligned to MN spike peaks. Ab and Bb, histograms of spike-burst delays, i.e. interval between spike peak and the nearest centre of gravity point of VR burst in each cycle. Each histogram displays all cycles from one complete episode in each condition. Ca, normalized spike-burst delay of time control and 18β-GA treatment experiments. The spike-burst delay was normalized by calculating Tdelay /PTdelay , where PTdelay and Tdelay are spike-burst delay of pre-treatment and treatment, respectively. Pre-treatment parts of each experiment were normalized to 1 and indicated by the dotted line. Cb, S.D. of the delay. Open bar: pre-treatment; filled bar: +30 min time control (n = 3) and 18β-GA (n = 7). ∗∗ P < 0.01; ∗∗∗ P < 0.001. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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not shown) had any significant effect on the duration of ventral root bursts (Fig. 9Ab lower trace and Ba) or on the duration of swim episodes (Fig. 9Bb) after equivalent exposure times to when applied at stage 37/38. Discussion The main conclusion from this study is that in hatchling Xenopus embryos electrical coupling synchronizes the firing during swimming of homonymous motoneurons innervating the myotomal muscles. The evidence derives from the fact that a consistent effect of gap junction blockers was an increase in the duration of rhythmic ventral root bursts during fictive swimming. In particular, low concentrations of 18β-GA (30 μM), which have few if any deleterious effects on the properties of neurons (Li et al. 2009), caused a significant increase in burst

Figure 7. The effects of 18β-GA (90 μM) on passive motoneuron properties Aa, the resting membrane potential in control and after treatment with 18β-GA. Open bars = time 0 (pre-treatment); filled bars = +30 min (either treatment or control). Ab, normalized numbers of PSPs during quiescent periods between swimming episodes. The treatment parts of each experiment were normalized by calculating TNpsps /PTNpsps , where PTNpsps and TNpsps are pre-treatment and treatment parts, respectively. The pre-treatment part was normalized to 1 and is indicated by the dotted line. B, 18β-GA effects on membrane input resistance. Ba, a hyperpolarizing current (−30 pA) was applied and the membrane responses were recorded pre-treatment (black trace) and after 18β-GA treatment (grey trace). Bb, averaged membrane input resistance of control and 18β-GA experiments. Open bar: pre-treatment; filled bar: time control (n = 3) or 18β-GA treatment (n = 7). ∗∗∗ P < 0.001. C 2009 The Authors. Journal compilation C 2009 The Physiological Society

Figure 8. The effects of 18β-GA (90 μM) on motoneuron firing properties Aa and b, spike responses to suprathreshold 110 pA depolarizing current pulses pre-treatment and after 18β-GA to show multiple spike capability in both situations. Ba, example of spikes induced by depolarizing pulse at threshold in control (black trace) and 18β-GA treatment (grey trace). Ca, average of 25 consecutive spikes from pre-treatment control (black trace) and 18β-GA treatment (grey trace) during fictive swimming. Bb and Cb, normalized spike widths in control saline (n = 3) and 18β-GA (n = 7) experiments. The treatment parts of each experiment were normalized by calculating Twidth /PTwidth , where PTwidth and Twidth are pre-treatment and treatment parts, respectively. The pre-treatment part was normalized to one and indicated by the dotted line. ∗∗ P < 0.01; ∗∗∗ P < 0.001.

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durations without affecting either swim episode durations or the frequency of fictive swimming. This increase in burst durations appears to result exclusively from a desychronization of single spike per cycle activity, rather than a change in the number of spikes that motoneurons discharge in each cycle of swimming. At higher concentrations, 18β-GA (90 μM) produced an even more dramatic increase in ventral root burst durations and also led to a large reduction in episode durations. This separation of effects on two key parameters of swimming could result from a differential gap junction block of motoneuron-to-motoneuron connections within the spinal cord, versus coupling between excitatory premotor interneurons in the hindbrain and rostral spinal cord, two loci in the motor system where electrical coupling is known to be prevalent (Perrins & Roberts, 1995a,b; Li et al. 2009). For example, electrical synapses between spinal motoneurons may be more susceptible to pharmacological blockers than those between neurons in the brainstem because the spinal cord is a much smaller piece of tissue. The main effect of gap junction block between spinal motoneurons might be expected to be an increase in burst duration if electrical coupling is important for synchronization of motoneuronal spikes. This outcome of gap junction blockade is similar to the effect of reducing electrical coupling strength between motoneurons reported in a recent semi-quantitative computer modelling study of the stage 37/38 swimming network (Wolf et al. 2009). A decrease in coupling between pre-motor interneurons might also contribute to this by de-synchronizing the excitatory drive to motoneurons. In addition, however, the excitatory interneurons play a critical role in maintaining swimming (Li et al. 2006), so that at higher concentrations of 18β-GA, when coupling between them is more strongly

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reduced, the ability of the system to maintain swimming activity itself is compromised, and episode durations are consequently reduced. Electrical coupling has also been found in different loci in the rodent spinal cord; between motoneurons, and between excitatory, locomotor-related Hb9 interneurons, which play different roles in the locomotion (Walton & Navarette, 1991; Chang et al. 1999; Hinckley & Ziskind-Conhaim, 2006; Wilson et al. 2005, 2007). 18β-GA had two associated effects on the integrative electrical properties of spinal motoneurons: an increased input resistance and an increased spike width. The increase in input resistance is explained by the closure of low resistance electrical synapses between neighbours, and has also been reported previously following block of gap junctions between neurons in the tadpole hindbrain (Li et al. 2009), in the mammalian inferior olive (Leznik & Llinas, 2005) and in smooth muscle cells that are members of an electrotonically coupled syncytium (Takeda et al. 2005). An increase in spike width following gap junction block with 18β-GA has also been reported in the inferior olive (Leznik & Llinas, 2005) and in tadpole hindbrain neurons (Li et al. 2009). In the inferior olive it was suggested that the contribution of a distal dendritic calcium current to the somatic voltage response increased due to the removal of the current sink effect of more proximally located gap junctions (Leznik & Llinas, 2005). Whether or not a similar explanation underlies the spike width change seen in the tadpole spinal cord remains to be determined. The effects of 18β-GA and CBNX on swimming are reminiscent of the changes that normally occur as early larval development progresses. By larval stage 42, ventral root bursts increase in duration compared to earlier stages, and the relationship between the cycle

Figure 9. Lack of effect of the gap junction blocker 18β-GA (90 μM) on fictive swimming in larvae Aa Stage 42 Xenopus larva (Nieuwkoop & Faber, 1956). Ab, excerpts of fictive swimming from near the start of an episode evoked by tail skin stimulation in control saline (upper trace) and 30 min after application of 18β-GA (lower trace). Note long control larval bursts are unaffected by 18β-GA and resemble stage 37/38 activity after gap junction block with 18β-GA (e.g. Figs 1Ab; 3Ab). B, neither ventral root burst duration (Ba) nor swimming episode duration (Bb) was significantly affected by 90 μM 18β-GA (n = 6). Normalized episode duration was calculated by TDur /PTDur , where PTDur and TDur are episode duration of pre-treatment and treatment, respectively. Open bars = time 0 (pre-treatment); filled bars = +30 min (18β-GA). C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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period and rostrocaudal delay switches to one in which these two parameters begin to correlate (Sillar et al. 1991, 1992; Tunstall & Sillar, 1993). As we show here, these two developmental changes in the parameters of swimming can similarly be induced in embryos following gap junction block by 18β-GA, raising the possibility that a decrease of electrical coupling is an important and necessary feature of locomotor network maturation in Xenopus tadpoles. Strong support for this hypothesis came from the finding that gap junction block at stage 42 has no detectable effect on fictive swimming, suggesting a developmental reduction in the role of electrical coupling in the coordination of locomotor activity. Although gap junction coupling plays an important role in certain areas of the adult vertebrate CNS, such as the retina (Dowling, 1991) and the hippocampus (Draguhn et al. 1998), in other areas including the neocortex and the spinal cord, gap junctions appear to play a more transient developmental role (reviewed in Caveney, 1985; Roerig & Feller, 2000). Thus, it is not without precedent that gap junction coupling between spinal neurons, notably in amphibians (Spitzer, 1982), but also in mammals (Walton & Navarette, 1991), declines as development proceeds. A decrease in electrical coupling may contribute significantly to motor system development in Xenopus tadpoles but it cannot account for it completely. The transition from brief to longer, more complex ventral root bursts during development from hatching stage 37/38 to larval stage 42 is also accompanied by an increase in the number of spikes per cycle fired by individual motoneurons (Sillar et al. 1992), but this does not happen at stage 37/38 in the presence of 18β-GA. Indeed it is known that stage 42 and stage 37/38 neurons differ in their complement of ion channels, and this in turn imposes changes in their discharge properties (Sun & Dale, 1998). If a decrease in electrical coupling is indeed an important contributory factor in the maturation of the Xenopus swimming system, then the question arises of what developmental signals normally trigger it. The presence at early stages of vertebrate embryonic development of a wide range of neurotransmitters and other signalling molecules suggests they may play important roles in synaptogenesis and circuit formation (Roerig & Feller, 2000). It is of considerable interest in the context of the present study that biogenic amines serotonin (Roerig & Sutor, 1996; Moss et al. 2005) and dopamine (Dowling, 1991) have been reported to down-regulate gap junctional coupling between neurons. Furthermore, several lines of evidence link serotonin to neural network development in tadpoles (Sillar et al. 1995) and in other species (Roerig & Sutor, 1996; Moss et al. 2005). The postembryonic development of the swimming rhythm in Xenopus tadpoles follows a rostrocaudal sequence, with the larval bursts appearing first in rostral regions at stage 40 before progressing to caudal segments by stage 42 C 2009 The Authors. Journal compilation C 2009 The Physiological Society

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(Sillar et al. 1991). This broadly parallels the timing of the innervation of the spinal cord by serotonergic fibres from the developing raphe region (van Mier et al. 1986). Moreover, there is evidence for a causal link between the two phenomena because embryonic ablation of raphe projections prevents the development of the characteristic larval swimming pattern (Sillar et al. 1995). The main effect of 18β-GA on fictive swimming at stage 37/38 is an increase in burst durations and this effect is distributed along the rostrocaudal neuraxis indicating that electrical coupling is present between motoneurons innverating segments at least as caudal as the 12th post-otic cleft. As in normal development (Sillar et al. 1991), the burst durations in 18β-GA are greater rostrally than caudally. In this way 18β-GA transforms the embryonic rhythm into one more closely resembling that recorded at later larval stages. This leads to the testable hypothesis that under normal circumstances serotonin release from raphespinal projections reduces gap junction coupling first in rostral segments and only later more caudally to confer the appropriate temporal dynamics on CPG development, a hypothesis that will be pursued in future studies. References Caveney S (1985). The role of gap junctions in development. Annu Rev Physiol 47, 319–335. Chang Q, Gonzalez M, Pinter MJ & Balice-Gordon RJ (1999). Gap junctional coupling and patterns of connexin expression among neonatal rat lumbar spinal motor neurons. J Neurosci 19, 10813–10828. Connors BW & Long MA (2004). Electrical synapses in the mammalian brain. Annu Rev Neurosci 27, 393–418. Davidson JS & Baumgarten IM (1988). Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships. J Pharmacol Exp Ther 246, 1104–1107. Dowling JE (1991). Retinal neuromodulation: the role of dopamine. Vis Neurosci 7, 87–97. Draguhn A, Traub RD, Schmitz D & Jefferys JGR (1998). Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro. Nature 394, 189–192. Fuentealba P, Crochet S, Timofeev I, Bazhenov M, Sejnowski TJ & Steriade M (2004). Experimental evidence and modelling studies support a synchronizing role for electrical coupling in the cat thalamic reticular neurons in vivo. Eur J Neurosci 20, 111–119. Hinckley CA & Ziskind-Conhaim L (2006). Electrical coupling between locomotor-related excitatory interneurons in the mammalian spinal cord. J Neurosci 26, 8477–8483. Kiehn O & Tresch MC (2002). Gap junctions and motor behaviour. Trends Neurosci 25, 108–115. Leg´endy CR & Salcman M (1985). Bursts and recurrences of bursts in the spike trains of spontaneously active striate cortex neurons. J Neurophysiol 53, 926–939. Leznik E & Llinas R (2005). Role of gap junctions in synchronized neuronal oscillations in the inferior olive. J Neurphysiol 94, 2447–2456.

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