Soffe SR, Roberts A, Li WC.

Wellcome Trust

	Figure 1. The hatchling Xenopus tadpole and adjustments to timing measurements according to recording sitesA, diagram of the tadpole in side view to show the CNS (grey) and its regions (f, m, h: fore-, mid- and hind-brain; s: spinal cord; oc: otic capsule;*: obex) with swimming muscles (sm) in chevron shaped segments. White dot indicates the neuron in B. B, diagram of the CNS and swimming muscles in dorsal view to show typical sites for electrodes to record from a single neuron (patch) and ventral roots on the ipsilateral (vri) or contralateral (vrc) side. Measured timings of neuron activity were adjusted as though relative to recordings from either an assumed, adjacent ventral root (vradj) or the 5th post-otic ventral root (vr5th) on the same side as the recorded neuron (arrows and grey electrodes; see text for details).
	Figure 2. Excitatory connections from dINsA, diagram of the CNS in dorsal view to show a recorded dIN and motoneuron (mn) (shown in B–D). B, photomicrograph of neurobiotin-filled neurons in lateral view where a rostral dIN with short dendrites has a descending axon that passes a more caudal motoneuron (mn) with mainly ventral dendrites and identified by its peripheral axon. C, the dIN in B shows a characteristic single, broad action potential in response to injected depolarising current. D, paired recording from the neurons in B (at two time scales; Mg2+ omitted from saline) shows that current-evoked spikes in the dIN produce long EPSPs in the motoneuron (left) at consistent, short latencies (right). E, latencies of EPSPs increase with separation between presynaptic dINs and their postsynaptic targets (dIN–dIN synapses, circles; dIN–non-dIN synapses, triangles). The regression equation is d = 1.04 + 2.79s where d is delay in ms and s is separation in mm.
	Figure 3. Activity of a reticulospinal dIN during swimmingA, anatomy of a recorded dIN with ascending and descending axons and mainly dorsal dendrites. The diagram shows the soma position and recording arrangement. The ventral root electrode (vr) was placed on the opposite side to the intracellular recording (black). B, single episode of swimming, monitored with the ventral root electrode (vr), showing the reliability of firing of a dIN. C, expanded swimming cycles from B to show the normal swimming pattern (*: IPSPs; arrowhead: spike). The grey vr trace shows the motor activity from the recorded, vr trace (black) adjusted to show the assumed timing of ipsilateral motor activity (as though recorded ipsilaterally by the grey electrode in diagram A). D, equivalent cycles of swimming recorded from the same dIN under voltage clamp reveal the underlying, rhythmic synaptic drive (*: IPSCs; arrowhead: fast EPSC). The grey vr trace is the assumed ipsilateral motor activity as in C.
	Figure 4. Spikes in dINs are early relative to those in non-dINs and vrs during swimmingA, overlapped spikes of a motoneuron (mn) and dIN aligned relative to the start of ‘adjacent’ ventral root (vr) bursts (dashed line) show that the dIN fires earlier. The inset diagram shows the typical recording arrangement (actual recording site and vr bursts, black; adjusted recording site and vr bursts, grey; see Fig. 1). B, distribution of spike peak timing for the two neurons in A. C, the relative timing of spike peaks for 16 dINs (dark bars) and 15 non-dINs (light bars), all adjusted to give timings relative to an adjacent ventral root. Distributions of spike timings for each neuron (e.g. B) were normalised (as % of the whole sample of spike times for each neuron, using 2 ms bins) and then combined to give overall spike time distributions for dINs and non-dINs.
	Figure 5. Distinguishing neurons by spike shape in loose patch recordingsA, locations of illustrated neurons (from separate recordings; contralateral ventral root electrode). B, anatomical characterisation of a dIN (upper micrograph) with 2 large dorsal dendrites and a single descending axon (an additional axon is from a separate neuron) and one of a pair of motoneurons (lower micrograph; mn1; dotted lines indicate displacement of axons during processing). Black and white arrowheads indicate the dorsal and ventral limit of the CNS respectively (h: hindbrain, s: spinal cord). C, loose patch recordings of activity from a non-dIN neuron (mn1) and a dIN during swimming, monitored from a ventral root on the opposite side. D, distinguishing dINs from non-dINs by extracellular spike shape (upper records, normalised to the amplitude of the initial peak; p: peak-to-peak amplitude, t: trough amplitude, w: width at half initial peak amplitude). Lower records are corresponding spikes during swimming after attaining the whole-cell configuration (all averages of ∼20 spikes). E, cluster analysis distinguishes two groups of neurons that match a separation based on anatomical features (circles, dIN; triangles, non-dIN; open symbols are the illustrated neurons). F, cluster analysis on a larger group of neurons (n= 52) selected at random from those used to measure spike timing during swimming (see Fig. 6) again separates the neurons into two groups, equivalent to the dINs and non-dINs in E.
	Figure 6. Timing of neuron spikes during swimmingA, timing of spikes in a dIN (upper) and non-dIN (lower) recorded in the same animals with loose patch, aligned relative to the start of vr bursts adjusted to the 5th post-otic cleft. The vertical dashed line indicates vr burst onset. B, the timing of spikes (±s.d.) in dIN (circles) and non-dIN (triangles) neurons recorded at different longitudinal positions in a single animal. C, the timing of spikes (±s.d.) at different longitudinal positions for the whole data set (103 neurons). The dorsal view of the CNS and swimming muscles (labelled in Fig. 1B) is shown at the same scale and horizontal alignment as the graph; the small lateral view of the tadpole shows the region illustrated (*: obex; grey arrow: 5th post-otic cleft used as a timing reference; horizontal dashed line: 0 ms). D, the timing of spikes as in C showing neuron identification (dIN, circles; non-dIN, triangles) and contiguous regression lines (0–0.725 mm: y= 9.575 – 20.605x, where y is in ms and x is in mm, R2= 0.51; from 0.725 mm: y=–13.013 + 10.659x; R2= 0.63). Approximate rhombomere boundaries and obex (*) are indicated. E, deviation of timing measurements from the regression lines in D (slope: 0.05 ms mm−1, R2= 0.00). F, mean firing times of dINs (dark bars) and non-dINs (light bars), transformed to remove the effect of longitudinal position as in part E.
	Figure 7. The relative timing of spikes and EPSCs during swimmingA, recording from a non-dIN neuron under voltage clamp during swimming showing EPSCs for timing measurement (e.g. box). Swimming was monitored from a ventral root on the opposite side (vr: shown adjusted as though recorded from the same side). B, superimposed, spikes and vr bursts from consecutive swimming cycles in a non-dIN and a dIN. Below each are overlapped EPSCs from separate swimming sequences in the same neurons, also aligned to the starts of vr bursts (adjusted as in A). C, the timing of spikes and EPSCs (median values from individual neurons) measured relative to vr burst onset (dIN, circles; non-dIN, triangles; line indicates equality). D, the delay between EPSCs and spikes in the same neuron, plotted for individual dINs (dark bars) and non-dINs (light bars). E, overlapped averages of spikes recorded from different neurons during swimming to show pre-potentials (= EPSPs) in non-dINs and immediate take-offs in dINs (arrows).
	Figure 8. Post-inhibitory rebound in dINsA, rebound spikes to current pulses (dIN1), then a rebound spike following a spontaneous IPSP (at arrow), producing an EPSC (*) in a postsynaptic neuron (dIN2). B, when held just above spike threshold, a dIN fires on rebound following an IPSP (at arrow) elicited by the second of two spikes evoked in an inhibitory interneuron (Inh IN) by depolarising current (bars). Evoked and rebound spikes in the dIN both evoke EPSPs (*) in the inhibitory interneuron. Right, averages of 5 rebound spikes in the dIN and resulting EPSPs in the inhibitory interneuron. C, rebound in a dIN, depolarised just above spike threshold, following IPSPs (arrow) evoked by stimulation (at arrowhead) of the contralateral side of the spinal cord (upper overlapped traces). No rebound occurs where IPSPs fail (middle traces) or when the dIN is at rest (lower traces).

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