Li WC , Roberts A , Soffe SR .

Wellcome Trust

	Figure 1. Xenopus tadpole, swimming CPG, and electrical coupling between dINs with their anatomyA, the tadpole swimming CPG comprises half-centres on each side of the spinal cord (squares). Each half-centre has populations of cINs, aINs, dINs and MNs (more details in text). Filled circle connections are inhibitory and triangles are excitatory. Connections onto a half-centre affect all neurons in it. Resistor symbols indicate electrical coupling between MNs and between dINs. The drawing (right) shows the tadpole at developmental stage 37/38. B–E, paired recording of two dINs filled by neurobiotin. B, when either dIN was hyperpolarised by square pulse current injection (bars, dIN1: −70 pA, dIN2: −100 pA), a smaller response was seen in the other neuron. C, the shape of spikes evoked at the start of a step current injection into one dIN and the responses in the other. dIN2 makes electrical synapse onto dIN1. In contrast, dIN2 displayed EPSPs (arrow) superimposed on the electrical synaptic response evoked by dIN1 firing. The EPSPs were consistent in latency but variable in size indicating a monosynaptic chemical connection. Five traces overlapped. D, high power tracing shows dIN1 has both ascending and descending axons (red, a1, d1) but dIN2 only has a descending axon (blue, d2). The descending axon of dIN1 may contact the dIN2 soma and basal dendrite (*). E, low power tracing shows somata and descending axons closely apposed to each other for a distance of about 200 μm (between arrowheads). Vertical dashed line indicates the hindbrain/midbrain border which is set as 0 in longitudinal position measurements.
	Figure 2. The properties of electrical coupling between pairs of dINsA, scatter plot of coupling coefficients. Negative-current test pulses were injected into the rostral dIN (R-C) or the caudal dIN (C-R). Regression: y= 1.7 + 0.91x (R2= 0.63, n= 55, P < 0.001). B, coupling coefficients (average of both directions for each dIN pair) decrease with the distance separating the two dIN somata (regression y= 10.3 − 0.03x, R2= 0.13, n= 31, P= 0.049). C, examples of responses of dIN2 to sinusoidal current injection into dIN1 at 3 and 30 Hz, respectively. D, the dependency of electrical coupling coefficient on the frequency of injected sinusoidal current (mean ±s.d., n= 5 dINs). E, paired recording showing that attenuation between electrically coupled dINs is greater for spikes than for sustained depolarisation. Injection of current (cur) into dIN2 depolarises the cell and evokes a single spike, producing a depolarisation and spikelet in the electrically coupled dIN1 (left and middle). Spikes in dIN1 produce similar spikelets in dIN2 (right). In this example there is no chemical synaptic interaction.
	Figure 3. Electrical coupling and dIN firing during swimmingA, recording of activity in a dIN during swimming, monitored with a ventral root recording on the same side (VR). The dIN fires reliably on each cycle. B, hyperpolarisation to reduce firing to ∼50% of cycles reveals underlying ‘on-cycle’ excitation (open arrowhead) whose peak does not even reach the resting potential (dotted line). C, at an extended time scale, impulses remaining during hyperpolarisation are delayed and can arise from the falling phase of the underlying excitation (e.g. red trace). D, swimming recorded in an aIN and a cIN where firing is occurring on ∼50% of cycles. Spikes fail while the underlying on-cycle excitation is still substantially above the resting potential. VR in cIN was recorded from the opposite side. E, estimates of firing threshold at rest (open bars) and during swimming (grey bars) for three interneuron types. Neuron numbers are indicated (**P < 0.001). F, a dIN again firing reliably during swimming; initiated by a short current pulse to the skin (*); monitored by VR on the opposite side. Reliable firing continues even with −380 pA current injected into the dIN. When the hyperpolarising current is increased to −420 pA, firing starts to fail and reveals the underlying on-cycle excitation on some cycles (open arrowheads), and all-or-none, probable axonal action potentials on others (filled arrowheads; see text). G, overlapped spikes from F, (synchronised to VR burst onset). Injection of −380 pA delays the spikes. Injection of −420 pA causes some spike failure revealing small, early-onset potentials (black = synaptic excitation: open arrowhead) and larger, later-onset potentials (red = axonal action potentials: filled arrowhead) which are delayed like the remaining full spikes (blue). Dotted lines represent the resting potential in all cases.
	Figure 5. The effect of 18-β-GA on dIN properties after the block had establishedA, a dIN's responses to positive and negative current injection in control and 40 min after 18-β-GA application. B, overlapped unitary EPSPs evoked by dIN firing in control and after the 18-β-GA coupling block had stabilised. The dotted traces are records where EPSP failed. Note the disappearance of the spikelet (Fig. 2E) in block. C, comparison of dIN properties in control and 18-β-GA block. AP is action potential and AHP is afterhyperpolarization (n= 16 dINs unless indicated). Error bars represent s.d.
	Figure 4. The effects of gap junction blockers on dIN properties 5–10 min after their applicationA, an example dIN pair where 200 μm FFA was applied. After 5 min the RMP (control level shown as a dotted line) became more negative in both dINs (arrowed) and there was a dramatic increase in SSP frequencies in dIN2. After 12 min in wash, both effects disappeared. There was a small increase in Rinp in both neurons. Current injection was −100 pA in dIN2 in FFA and Wash and −120 pA in both dINs in other cases. B, summary of effects of four gap junction blockers on RMP, spike height, Rinp and SSP frequency (measured when RMP was hyperpolarized and all synaptic potentials appear depolarizing) within 5–10 min of blocker application. Numbers in brackets are the number of dINs analyzed for each measurement. Significance is indicated at P < 0.05 (*) or P < 0.01 (**; paired t-test). Open bars are control. Grey bar measurements were made 5–10 min after application of heptanol (Hep; 2 mm), carbenoxolone (Carb; 100–300 μm), FFA (100–400 μm), or 18-β-GA (40–60 μm). Error bars represent s.d.
	Figure 6. Time course of electrical coupling block by 18-β-GA (40 μM; applied at time = 0) and swimming pattern changesA, determination of the time course of coupling block by 18-β-GA in a single example. Data points were chosen to give near-horizontal regression lines at the start and end (open circles: y=−0.0007x+ 96.827, R2= 0.0005, and filled circles: y=−0.0012x+ 22.65, R2= 0.0044, respectively). Data points in the middle as block progresses were fitted with a third line (grey circles: y=−0.0573x+ 142.93, R2= 0.9496). The times when block starts (13.4 min) and stabilises (35.9 min) are given by the intersection of regression lines (see text for more details). B, time series of normalised electrical coupling strength measurements in 6 dIN pairs. Grey shading is between the mean times for the block to be detected and to reach stable block, respectively. C, time series of normalised swimming cycle period and episode lengths in the 6 paired dIN recordings. Grey swimming plots are data for individual recordings and black ones are averaged measurements (mean ±s.d.).
	Figure 7. The reliability of dIN firing following 18-β-GA applicationA, 2 dINs show typical, reliable, 1 spike per cycle firing in swimming before 18-β-GA application. Swimming was started by electrical stimulation on the skin (*) and lasted for 93 s (grey bars show the break in recording). There is a typical swimming frequency drop from the beginning to the end of the episode. B, while the coupling block is progressing (44% of control), dIN1 only fires 1 spike while dIN2 still fires on the majority of swimming cycles. Swimming was spontaneous and just the start (dots indicate a brief sampling gap) and the end are shown (grey bars show the break in recording). C, when the 18-β-GA block is stable (12% of control), the swimming episode (started by skin stimulation at *) is shortened to ∼1.3 s. dIN1 fires 3 spikes while dIN2 still fires very reliably. D, summary of the changes in firing reliability of 16 dINs in 9 animals before block and in final 18-β-GA block. Lines indicate changes in individual neurons. E, time series plots of firing reliability (%) for 12 dINs in 6 paired recordings. Grey curves and symbols in the upper panel are 6 dINs which fired reliably throughout 18-β-GA application. Reliability for 6 dINs in the lower panel dropped gradually and then stayed at low levels. Filled symbols show averaged time series of firing reliability of all 12 dINs (mean ±s.d.). The time for the episodes illustrated in A, B and C is marked in E (open circles).
	Figure 8. The firing of dINs in paired recordings became less synchronous when the electrical coupling was blockedA, examples of less synchronous dIN firing in 18-β-GA. dIN spikes in 10 consecutive swimming cycles were lined up and superimposed to dIN2 spikes in control before 18-β-GA application and at electrical coupling block. In control, differences in timing are relatively small (range arrowed) and dIN1 firing is consistently earlier than dIN2. During coupling block, differences in timing are larger and some dIN1 spikes are later than dIN2 spikes. B, anatomy of the dINs in A. Diagram shows the location of dINs in caudal hindbrain/rostral spinal cord (sc). Dorsal is upwards. The photograph shows dIN anatomy in more detail: dIN1 has an ascending axon (a1) and a descending axon (d1) which contacts the basal dendrites of dIN2 (*) where electrical coupling may take place; dIN2 has only a descending axon (d2).
	Figure 9. Membrane potential trajectories of dINs in swimming lose their similarity during 18-β-GA applicationA, simultaneous recordings from 2 dINs (red and black traces overlain) firing reliable spikes throughout 18-β-GA application. Membrane trajectories are very similar in control. After electrical coupling block in 18-β-GA, this similarity is reduced. B, the cross-correlation (cc) between dIN recordings in A (black = control; blue = 18-β-GA). C, a second example of a dIN paired recording and D, its cross-correlation, where only one dIN fires reliable spikes during coupling block. The silent dIN (red trace) receives weak synaptic drive during coupling block. E, time series plots of cross-correlation peak values (cc peak) for 6 dIN pairs during swimming. Two dIN pairs continued to fire reliable spikes throughout 18-β-GA application; their correlation peak values change relatively little (top). In the other four pairs, one or both of the pair stopped firing reliably; their correlation peak values fall during 18-β-GA application (bottom). F, time series plot of cross-correlation lags; these change little during 18-β-GA application.
	Figure 10. Summary of time series measurementsValues (mean ±s.d.) are for electrical coupling, dIN activities and swimming parameters in six paired recordings. Grey shading is between the mean times for the block to be detected and to reach stable block, respectively (see Fig. 6B).

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