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The Journal of Neuroscience, February 15, 1998, 18(4):1602-1612
The Pharmacology and Roles of two K+ Channels in Motor Pattern Generation in the Xenopus Embryo
Frederick M. Kuenzi and Nicholas Dale School of Biomedical Sciences, University of St. Andrews, St. Andrews, Fife KY16 9TS, United Kingdom
ABSTRACT
Top
Abstract
Introduction
The spinal neurons of the Xenopus embryo that participate in the swimming motor pattern possess two kinetically distinct sets of potassium currents: the fast IKf and sodium-dependent IKNa, which together constitute ~80% of the outward current; and the slow IKs, which constitutes the remainder. To study their respective roles in cell excitability and the swimming pattern, we have characterized their pharmacological properties. Catechol selectively blocked the fast potassium currents (IC50, ~10 µM). The block was voltage-dependent, with partial unblocking occurring at positive voltages.
-Dendrotoxin and dendrotoxin-I selectively blocked the slow potassium current. Catechol and the dendrotoxins had different effects on membrane excitability: catechol caused spike broadening but had little effect on repetitive firing, whereas both dendrotoxins markedly increased repetitive firing without affecting spike width. By applying these agents to the whole embryo, we tested the role of the fast and slow currents in motor pattern generation. Catechol had little effect on fictive swimming, suggesting that the fast K+ currents are not critical to circuit operation. However, dendrotoxin disrupted swimming early in the episode and increased the duration of ventral root bursts. The slow K+ current, which is a minor component of the total outward current, thus appears to play an important role in motor pattern generation.
Key words: potassium channels; catechol; dendrotoxin; 4-aminopyridine; Xenopus; central pattern generator; neural model; repetitive firing
INTRODUCTION
The pattern of neural output produced by a circuit of interconnected neurons depends on the biophysical properties of the constituent neurons, the pattern of synaptic interconnections, the transmitter-receptor systems of those synapses, and the neurohumoral environment. The roles of individual voltage- and ion-gated ion channels have been studied in shaping reflex responses (Byrne, 1979
), and recently this analysis has been extended to more complex circuits (e.g., Golowasch and Marder, 1992
The Xenopus embryo is a preparation in which detailed understanding of how ion channels contribute to motor behavior is possible. The embryonic neurons that control swimming fire a single spike on each cycle. Discharge on either side of the body alternates, causing flexions that drive the animal forward. The spinal circuitry that generates swimming has been well characterized (Arshavsky et al., 1993
We have identified pharmacological agents that selectively block the fast and slow potassium currents in Xenopus spinal neurons; catechol specifically inhibited IKf and IKNa in a voltage-dependent manner, whereas both dendrotoxin-I (DTX-I) and
MATERIALS AND METHODS
Dissociation of spinal neurons. Spinal neurons of stage 37/38 Xenopus laevis (Nieuwkoop and Faber, 1956
Cell identification. Neurons that had been acutely dissociated in this way retained much of their morphology and could be identified by the criteria of Dale (1991)
Patch recording in isolated cells. For making whole-cell patch recordings the normal solution bathing the cells was exchanged for a "control" recording medium that contained (in mM) 115 NaCl, 3 KCl, 1 MgCl2, 10 CaCl2, 2.4 NaHCO3, and 10 HEPES. The pH was adjusted to 7.4, and the osmolarity was ~260 mOsm. The intracellular solution contained (in mM) 100 potassium methane sulfonate, 5 KCl, 6 MgCl2, 5 ATP (sodium salt), 2 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Molecular Probes, Eugene, OR), and 20 HEPES. The pH was adjusted to 7.4 with KOH, and the osmolarity was adjusted to 240 mOsm, usually by increasing the volume by 10%. Both of these solutions were sterile-filtered before use. In the dendrotoxin experiments we found that the block was more reliable if the extracellular calcium was reduced to 5 mM and the osmolarity was adjusted with glucose. The Ag/AgCl2 ground electrode was separated from the bath by an agar bridge containing control saline. Drugs were applied through a nine-barrel microperfusion apparatus with the nozzle positioned within 150 µm of the cell. Adequate flow was judged by the movement of cells and debris within the field of the microscope when the flow was turned on and off. In voltage-clamp experiments all solutions contained 150 nM tetrodotoxin to block sodium channels and 100 µM CdCl2 to block calcium currents (both from Sigma), and in all experiments the holding potential was
50 mV. In some experiments the sodium-dependent potassium current was blocked by replacing the sodium in the control medium with equimolar N-methyl-D-glucamine (Sigma). To measure INa we modified the external saline by replacing half of the NaCl with TEA-Cl and adding 100 µM CdCl2 and 1 mM 4-aminopyridine (4-AP). The internal saline contained (in mM) 100 cesium methane sulfonate, 1 CaCl2, 10 EGTA, 20 HEPES, 5 ATP, and 6 MgCl2, pH 7.4.
Recordings were made with a List Biologic (Campbell, CA) L/M EPC7 patch-clamp amplifier with the stimulus input and data acquisition controlled by an IBM-compatible computer and a Data Translation DT2831 data acquisition board. When filled with intracellular recording saline the electrode resistance was 4-10 M
in the bath. After breaking through to whole-cell configuration the series conductance was
0.1 µS, and in voltage clamp the series resistance compensation was typically between 75 and 80%.
Patch recording in the intact spinal cord. Although many neurons fired repetitively in cell culture, we found that repetitive firing was more reliable when making patch recordings in situ. In accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986, embryos were anesthetized with MS-222, and their dorsal fins were slit open using fine tungsten needles. They were incubated in 0.077 mg/ml
Drugs. DTX-I and
Fictive swimming. For convenience, in this paper fictive swimming recorded from the ventral roots of a paralyzed embryo will be referred to simply as "swimming." The preparation for extracellular recording of ventral root activity was similar to that described for in situ patch recording, except that the embryo was spinalized at the level of the first or second postotic myotome. The embryo was held upright between two pairs of tungsten pins so that glass suction electrodes could be pressed against the intermyotomal clefts to make ventral root recordings. Swimming was started by a brief electrical stimulus to the skin of the tail. At least 3 min elapsed between the end of the last swim episode and the next stimulus. Data were recorded on a cassette tape for later analysis.
To measure burst duration and cycle period, episodes of swimming were acquired with a Data Translation DT31EZ board onto a computer disk. The ventral root records were rectified and integrated over a 2 msec interval. Cycle period was calculated from the midpoints of the ventral root bursts, and the duration of a burst was measured as the time between the first crossing above a threshold and the last crossing below the threshold per burst. Thirty cycles were also averaged, triggered from the leading edge of the burst. The area under the averaged record was a measure of the number of spikes contributing to the bursts.
Statistical analysis. Results are presented as mean ± SEM. In dose-response curves the error bars are 1 SD. Exponential curves were fitted to experimental data using the Simplex algorithm, and concentration-response curves were fitted by the Levenburg-Marquardt method of least squares fitting (Press et al., 1988
RESULTS
Central pattern generator neurons and Rohon Beard neurons have different K+ currents
All of the cell types in this study possessed K+ currents that were not inactivated at a holding potential of
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Figure 1. The potassium currents of CPG neurons are faster than RB sensory neurons. A1, From a holding potential of
The channel kinetics were similar across cell types that make up the central pattern generator for swimming but were slower in the Rohon Beard sensory neurons. We separated the time constants from fits of a sample of 71 cells according to cell morphology (Fig. 1B,C; see Materials and Methods, Cell identification). An ANOVA including all cell types was highly significant (F(3,67) = 22.89 fast and 18.24 slow; p < 0.001 for both). In the planned comparison of time constants from the different (CPG) cell classes (Fig. 1B,C, Co, Mo, Mu) there was no difference for either the fast components or IKs between the different morphological types (F(2,46) = 0.06 fast and 0.42 slow). However, when the CPG groups were pooled and compared with RB neurons, the RB neurons were significantly slower for both components (F(1,69) = 70.57 fast and 55.27 slow; p < 0.001 for both).
A second difference between the cell types is in the fraction of the total outward current carried by IKs. By extrapolating the curves fitted to the tail currents back to the moment of repolarization, the amplitudes of the fast (IKf and IKNa) and slow (IKs) components could be estimated. Using this protocol 22 ± 2% of the tail current was IKs in CPG neurons compared with 40 ± 2% in RB cells (n = 49 CPG and 38 RB cells; p < 0.001). The similarity of the potassium currents across the CPG classes suggests that they are a homogeneous population and are quite distinct from the RB neurons. In addition, the pharmacological properties of RB K+ channels differed from those of CPG neurons (F. M. Kuenzi and N. Dale, unpublished results). Thus, we will not consider the RB neurons further in this paper. Only the properties of K+ currents in CPG neurons will be described.
Catechol specifically blocks the fast potassium currents
Catechol caused a rapid and reversible block of the K+ current at voltages lower than +20 mV. At voltages more positive than +20 mV the block partially reversed with a slow time course relative to channel opening.
During test steps to +10 or +20 mV the greatest block of the total current occurred early in the voltage step (Fig. 2A). The dose-response relationship for the percent block of the peak current early in the step (Fig. 2B, measured at the arrowhead in Fig. 2A) was well fitted by the Hill equation with an IC50 of 9.1 ± 1.9 µM and assuming a one-to-one binding (Hill coefficient = 1). When the Hill coefficient was allowed to vary, the best fit was 0.793 ± 0.162, which was not significantly different from 1, and the IC50 was not significantly different from the result using the simpler equation. The maximum block was 85.0 ± 3.6%, which corresponds well with the relative amount of fast current in these cells. The activation of the current became slower between 10 and 100 µM, which again suggested that the slow component was resistant to catechol. The current appeared to reach equilibrium faster in 1 mM than in 100 µM, which may be attributable to voltage-dependent unblocking at the lower concentrations (see below).
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Figure 2. Catechol selectively blocked the fast voltage-gated potassium currents. A, Effect of catechol on currents during the step recorded in control saline and the concentrations of catechol indicated. The voltage (top trace) was stepped from a holding potential of
We examined the tail currents to see whether catechol specifically blocked the fast component. Increasing the concentration of catechol to 100 and 1000 µM reduced the fast component but had little effect on the slow component (Fig. 2C,D). Paired comparisons of the percent block at each concentration of catechol showed that the fast component was blocked significantly more than the slow component at all concentrations. The dose response for the fast tail current was very similar to that of the total current (Fig. 2B). Again, the values in Figure 2D include partial unblocking during the test pulse (see Fig. 3B).
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Figure 3. Voltage dependence of catechol- resistant and -sensitive current. Example of total current in control (A1) and 100 µM catechol (A2). A3, Difference currents, obtained by subtracting traces in A2 from the corresponding ones in A1, show a decay in the catechol-sensitive current at positive voltages. Test voltages ranged from
The near total blockade of the fast component suggested that both IKf and IKNa are sensitive to catechol. We tested this directly by applying catechol in the presence and absence of external sodium. Replacing sodium in the control medium with N-methyl-D-glucamine reduced the total current by 31 ± 3%. This was the percentage of IKNa in these cells (Dale, 1993
In keeping with the effect on tail currents, the current that remained in 100 µM catechol activated with a slow time course across the physiological range of voltages (Fig. 3A2). Difference currents obtained by subtracting the current in 100 µM catechol from the control current (Fig. 3A1) at each voltage step showed a fast-activating, partially inactivating current (Fig. 3A3). At voltages of +20 mV and more, the current in catechol was greater than expected from the dose-response curve measured at lower voltages; it increased to ~50% of control rather than 20-30% predicted from Figure 2. This suggested that the block was voltage-dependent.
We constructed voltage-activation curves from tail currents after long voltage pulses. The fast and slow components were estimated by extrapolating the fitted curves back to the end of the test pulse as before. These curves showed that catechol appeared to shift the activation of the fast current toward more positive voltages (Fig. 3B). In six CPG cells the voltage for half-activation was 6.9 ± 1.6 mV in control and 26.4 ± 3.4 mV in 100 µM catechol (pairwise p < 0.01). In the four cells with a measurable slow component the voltage-activation curve was not shifted significantly (Fig. 3C).
Time dependence of catechol block
We explored the time and voltage dependence of catechol block further. The process of unblocking was studied by delivering a positive prepulse of varying duration and measuring the tail currents when the membrane was returned to
= 6.3 ± 1.1 and 5.5 ± 0.8 msec in control and catechol, respectively; n = 3) (Fig. 4C).
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Figure 4. The fast current showed time-dependent unblocking and reblocking. A, The pulse length of a voltage step to 20 mV was increased from 1 to 15 msec in control (A1) and 100 µM catechol (A2). The tail current was fitted with a double exponential to measure the fast (B) and slow (C) components in the two conditions (symbols as in A). Points were fitted by the equation I = Imax[1
Returning the cell to a negative voltage reverses this process and favors the reblocking reaction. We therefore measured the time dependence of reblocking by using a twin-pulse protocol (Fig. 4D). In the control, the activation of the current was the same in both pulses at all interpulse intervals, but in 100 µM catechol (Fig. 4D) the current during the second pulse activated much faster than the first at short interpulse intervals. As the interval increased the activation of the second pulse slowed and became more like the first, indicating a recovery of block. This recovery of block was examined in three cells by measuring the current 10 msec after the start of both pulses. The difference between the current during the first and second pulses at this point indicates the amount of unblocked current under these conditions (Fig. 4E). The points were well fitted by a single exponential, giving a time constant for reblocking of 141 ± 10.1 msec. Thus, IKf and IKNa undergo rapid, partial unblocking during the voltage step (Fig. 4B) followed by slow reblocking at the holding potential.
4-Aminopyridine preferentially blocks the fast current
In many types of neuron 4-AP specifically blocks the transient potassium current IA; however, we found that in embryonic Xenopus spinal neurons it blocked the sustained currents at low concentrations. With repeated pulses the block developed slowly, requiring >20 sec to reach a steady state, and it was only partially reversible. Like catechol, 4-AP slowed the activation of the current during the step (Fig. 5A). The concentration dependence of block for the current early in the pulse was also similar to that of the block by catechol (Fig. 5B). Assuming a single binding site, the data were well fitted by the Hill equation, with an IC50 of 15 ± 12 µM and maximum block of 82 ± 10%. There was a wide range of responses, with 100 µM causing near complete block (>80%) in some cells and <50% block in others.
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Figure 5. 4-Aminopyridine blocked both fast and slow potassium currents. A, During the voltage step the current during the beginning of the pulse is blocked more than the end. B, Concentration-response curve for the current early in the step (arrowhead in A). Error bars indicate 1 SD (n = 4-17 cells for each point). C, Tail currents at
Analysis of the tail currents showed that 4-AP preferentially blocked the fast component (Fig. 5C). In contrast to the effect of catechol, these concentrations also significantly reduced the slow component (p < 0.01 at 100 µM and p < 0.05 at 1 mM). Nevertheless, at every concentration the mean block of the fast component was greater than that of the slow component, and the pairwise differences in block were significant (Fig. 5D). The poor selectivity of 4-AP rendered it of limited value for functional studies, so it was not considered further.
Dendrotoxin-I and
DTX-I blocked the slow component of tail currents by ~40% at 0.5 µM (Fig. 6A,B). The slow component was blocked more than the fast component in a pairwise comparison (Fig. 6B), although the fast component was reduced by a small, but significant, amount. There was no measurable increase in the block of IKs at the highest concentration tested, 4 µM, nor was the selectivity less. In keeping with the relative size of IKs, the current during the step was reduced by only 10-15%.
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Figure 6. Dendrotoxin-I and
Dendrotoxin causes a small reduction in the sodium current
DTX has been found to modulate INa as well as blocking Ik in some preparations (Li and McArdle, 1993
Catechol broadens the action potential
The specificity of catechol and DTX allowed us to test the control that the fast and slow potassium currents have over neuronal firing properties. Whole-cell recordings were made under current clamp to measure changes in action potential duration and repetitive firing. To study the repolarizing phase of the action potential, a brief outward current pulse was delivered to a cell to initiate an action potential near the end of the pulse (Fig. 7A). We measured spike width as the time required for the voltage to return from the peak to one-third of the spike amplitude (resting potential to peak). In the control, spike width was 1.3 ± 0.1 msec; catechol increased this to 2.7 ± 0.6 msec at 100 µM (n = 10) and 4.1 ± 0.8 msec at 1 mM (n = 10; Fig. 7A). Both increases were significant at the 5% level. In a similar protocol and in repetitive firing studies (see below) the dendrotoxins caused no change in the spike width (mean width at 0 mV, 1.3 ± 0.1 and 1.2 ± 0.1 msec in control and 1 µM DTX, respectively; n = 5 (Fig. 8A-C).
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Figure 7. Catechol broadened spikes and slightly inhibited repetitive firing. A, In current-clamp recordings from a dissociated neuron a brief outward current pulse triggered a spike (bottom traces, voltage). One hundred micromolar and 1 mM catechol progressively slowed the repolarizing phase, broadening the spike compared with control. B, Repetitive firing of a cell that was depolarized by 80 pA to elicit a train of spikes in control saline. Ten micromolar and 100 µM catechol reduced the average frequency of firing and thus the number of spikes during the pulse. In this cell the initial frequency was also slightly reduced, but across all cells the change was not significant. Similar results were seen in cells that exhibited stronger accommodation.
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Figure 8. Dendrotoxin has little effect on spike width and increases repetitive firing. A, B, Current-clamp recordings from a dissociated cell (A) and a cell recorded in situ in control (A1, B1) and 1 µM DTX-I (A2, B2). After perfusing with 1 µM DTX-I the cells increased their firing, and the first spike was the same width as in control. C, A series of pulses of increasing magnitude were delivered to the cell in B to show changes in threshold and repetitive firing properties in DTX. DTX-I (filled circles) increased the number of spikes fired at each level of current. D, For three cells with similar thresholds the initial firing frequency (1/first interspike interval) is plotted against the current normalized to the control threshold current. At every current level the frequency in DTX (filled circles) was higher than in control (open circles).
Repetitive firing is slightly depressed by catechol but enhanced by dendrotoxins
Repetitive firing was examined by injecting a series of long outward current pulses of increasing amplitude, the highest of which was strong enough to activate multiple action potentials in most cells (Figs. 7B, 8B). In this study we found that the firing properties of cells ranged from those that had a graded response (number of spikes and frequency) to the amount of current injected to those that showed moderate or strong accommodation. Despite this variability in control conditions, the changes in firing caused by catechol and DTX were similar across cells. We considered changes in the total number of spikes and the threshold. Because the activity of cells during swimming suggested that the firing of the first two spikes was the most functionally relevant repetitive firing characteristic, we also measured the initial frequency (inverse of first interspike interval) at the current level that evoked at least two spikes in control. Both 10 and 100 µM reduced the total number of spikes during the train, but the change was only significant in 100 µM (p < 0.05; n = 7) (Fig. 7B). Catechol also lowered the threshold for firing spikes by 20 ± 8% at 10 µM (p < 0.05). The initial frequency, however, was not affected significantly by either concentration (n = 8 for 10 µM; n = 7 for 100 µM).
Dendrotoxins increased the repetitive firing capabilities of the cells. One micromolar DTX-I reduced the membrane accommodation, and neurons became capable of firing many more spikes in response to the same current injection (n = 10 of 12; Fig. 8A,B). An example of the relationship between current injected and the number of spikes evoked is shown in Figure 8C, and a similar trend was evident in five of six cells tested with graded steps. The threshold current decreased in 1 µM DTX-I to 57 ± 9% of control (p < 0.01; n = 6). In DTX the cells fired at higher frequencies at each current level. This is shown for three cells that had very similar thresholds (between 10 and 40 pA) in Figure 8D. At twice threshold current the frequency in control corresponded to an interspike interval of 20 ± 1 msec, which was significantly longer than the interspike interval in DTX-I (17 ± 1 msec; p < 0.05; n = 3). There were no noticeable differences between the effects of DTX-I and
The swimming pattern generator is very sensitive to changes in the slow current
We tested whether catechol block of the fast K+ currents could disrupt the basic pattern of swimming recorded in the ventral roots of the spinal cord. In control, the swimming pattern is characterized by brief bursts of activity that alternate on the two sides of the body; a burst on the right occurs midcycle of the burst on the left (Fig. 9A). The cycle period ranges from ~50 msec at the beginning of an episode to ~100 msec just before swimming stops. In the presence of 10 µM catechol, the basic pattern (cycle period, burst duration, and alternation) was unchanged (Fig. 9A, Table 1). After blocking at least 70% of the fast current with 100 µM, the embryo was surprisingly still capable of generating an alternating rhythm with a normal cycle period and burst duration. However, abnormalities were present, such as periods during which the ventral root discharge was missing on one side of the embryo (Fig. 9B). To examine the effects on burst duration more closely, the rectified and integrated extracellular record was averaged over 30 cycles in control (Fig. 10, thick line) and in catechol (Fig. 10, thin line). Ten and 100 µM catechol did not significantly change the number of spikes fired by motor neurons, as measured by the area under the curve (Fig. 10; change in area,
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Figure 9. The swimming pattern is relatively insensitive to catechol. Records of ventral root activity on the two sides of the embryo (vrl, vrr, left and right ventral root records, respectively) show that 10 µM catechol (A), which is near the IC50 for block of the fast current, has little effect on swimming; 100 µM catechol (B) caused some abnormalities in the pattern, with discharge absent on some cycles (*). Segments illustrated are taken 8 sec from the electrical skin stimulus that initiated the swimming episode.
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Table 1. Effect of potassium channel blockers on the swimming pattern (mean change ± SEM)
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Figure 10. Catechol has no effect, but DTX increases the burst duration during swimming. Ventral root recordings were rectified, integrated (2 msec steps), and averaged over the first 30 cycles. In each panel the thick line is in control, and the thin line is in the treatment indicated. Although catechol had no consistent effect on burst shape, DTX increased burst duration and increased the burst intensity (area under the curves).
In contrast to the relative insensitivity of motor pattern generation to catechol, the spinal circuit was sensitive to both
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Figure 11. Dendrotoxins disrupt the swimming pattern. A, Ventral root records from one side at the beginning and near the end of a swimming episode in control saline and
DISCUSSION
Pharmacological dissection of the fast and slow currents
Our results support the hypothesis that slow and fast potassium channels are distinct at the molecular level. In addition to having widely different opening and closing kinetics (Dale, 1995a
subunits in the native channels (Scott et al., 1994
For pharmacological agents to be useful in studying how ion channels influence circuit operation, they must satisfy two criteria: specificity for blocking one or a restricted set of channels, and maintenance of block during circuit operation. Catechol and DTX appear to satisfy both of these criteria in the Xenopus embryo; catechol blocks the fast currents, whereas DTX blocks the slow currents. Although we have demonstrated specificity for these two types of current, some stage 37/38 Xenopus neurons possess a small IA (F. M. Kuenzi and N. Dale, unpublished observations), and an "A" current is present in neurons grown in culture (Ribera and Spitzer, 1990
The second criterion, that the block is maintained during circuit activity (swimming), depends on the level of depolarization during activity within the circuit. During swimming the membrane potential is tonically depolarized to approximately
Kinetically distinct potassium currents play different roles in neural circuit function
The model of the Xenopus swimming circuit produced by Dale (1995b)
The model predicts that the swimming circuit is not very sensitive to changes in IKf but depends very strongly on IKs. Our results with catechol and DTX support this prediction; reducing the fast currents by 50% had no effect, whereas the block of IKs by DTX significantly altered the swimming pattern. In the model a 50% reduction of IKs did not disrupt the alternation between the two sides, but it did cause cells to fire a pair rather than a single spike in each cycle. In real embryos after treatment with DTX the alternation of ventral root activity on the two sides during swimming was normal. However, the ventral root burst duration was greatly increased. This increase could occur either through motor neurons firing a second spike during the excitatory phase of the cycle or through desynchronization of the motorneuron firing. Our data support the first interpretation. DTX increased the area under the curve of the averaged ventral root bursts (Fig. 10C). Because there was no spike broadening, more motor neuron spikes must have occurred during each cycle. This eliminates simple desynchronization, unless recruitment from a large pool of previously silent motor neurons occurred to provide extra spikes. This is unlikely because intracellular recordings from ventral neurons (primarily motor neurons) suggest that most fire during every swimming cycle (Soffe, 1993
Taken together these results confirm the predictions from our earlier model and suggest that IKs is a "strategic" current in the Xenopus swimming circuit. It is small in comparison with other outward currents (20% of the total) yet plays an important role in the function of the circuit. As such, the slow current could be a key target for modulation of the whole circuit. In vertebrate neurons some delayed rectifier currents are controlled by neuromodulators (Rehm and Tempel, 1991
Developmental changes in expression of IKs may also underlie changes in the swimming motor pattern that occur in the transition from embryo (stage 37/38) to larva (stage 41). During this period the pattern matures from the short bursts of ventral root activity in the embryo to much longer bursts in the larva (Sillar et al., 1991
FOOTNOTES Received Sept. 29, 1997; revised Nov. 25, 1997; accepted Dec. 2, 1997.
We thank the Biotechnology and Biological Sciences Research Council and the Royal Society for their generous support. We also thank Dr. William J. Heitler for his helpful comments on this manuscript.
Correspondence should be addressed to Nicholas Dale, Bute Medical Building, Westburn Lane, St. Andrews, Fife KY16 9TS, UK.
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